Methods and compositions for targeting progenitor cell lines

ABSTRACT

The invention provides methods, compositions and kits for the identification and enrichment of progenitor cell lines obtained from pluripotent stem cells.

This application claims priority to U.S. Provisional Application No.61/769,119 filed on Feb. 25, 2013 and to U.S. Provisional ApplicationNo. 61/692,139 filed on Aug. 22, 2012 both of which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The field of the invention relates to progenitor cells derived frompluripotent stem cells.

BACKGROUND

Pluripotent stem cells (PS), such as human pluripotent stem (hPS) cells,are capable of immortal proliferation in vitro and differentiation intoderivatives of all three embryonic germ layers (Cohen D E, Melton D(2011) Nat Rev Genet 12: 243). As a result, the isolation of hPS cells,which include human embryonic stem (hES) cells and induced pluripotentstem (iPS) cells (Blanpain et al. (2012) Nat Rev Mol Cell Biol 13: 471),has spurred new avenues of research to evaluate their potential toprovide a renewable source of human cells for basic research and asreplacement cells for the treatment of injury, aging, or any one of anumber of intractable degenerative diseases such as osteoarthritis,cardiovascular disease, macular degeneration, Parkinson's and perhapseven Alzheimer' s disease (Cohen D E, Melton D (2011) Nat Rev Genet 12:243; Blanpain et al. (2012) Nat Rev Mol Cell Biol 13: 471).Reprogramming methods for creating iPS cells from somatic cells(Nakagawa (2010) Adv Exp Med Biol 695: 215) have greatly expanded thenumber and diversity of PS cell lines, including hPS cell lines,available for research. Donor-derived hPS cells are a source of patientmatched cell types for disease modeling (Tiscornia et al. (2011) Nat Med17: 1570), drug screening (Laposa (2011) J Cardiovasc Pharmacol 58:240), and the development of potential autologous cell replacementtherapies (Nelson et al. (2010) Stem Cells Cloning 3: 29). However,there remains a need for efficient directed differentiation methods andimproved cell purification technologies for deriving various cell typeswith sufficient purity and known identity to meet the standards requiredfor translation into routine clinical application.

Current directed differentiation methods for obtaining specific maturecell types from hPS cells are sometimes limited by low efficiencies ofreproducibly yielding the desired cell types, and in certain instances,such preparations rarely exceed 30% purity (Cohen, Melton (2011) Nat RevGenet 12: 243). One approach to increasing the yield is enrichment ofdesired cell types using one or more progenitor-specific markers. Forexample, cell enrichment using surface antigens that define progenitorpopulations has been used to improve the yield of the desired cell typessuch as neural and cardiomyocyte progenitors (Dubois et al. (2011) NatBiotechnol 29: 1011; Yuan et al. (2011) PLoS One 6: e17540). Progenitorsurface markers could also be useful for monitoring and validating hPSdifferentiation and for high throughput screening of reagents thatstimulate differentiation toward a given lineage. However, apart frommapped hematopoietic progenitor markers, there is a paucity of validatedcell surface antigens for most embryonic progenitor cell lineages.

Phage display is a powerful ligand selection method that has beenapplied both in vitro and in vivo for the identification ofcell-specific targeting peptides (Molek et al. (2011) Molecules 16: 857;Teesalu et al. (2012) Methods Enzymol 503: 35). Peptide librariesdisplayed on phage particles are selected by repeated rounds ofenrichment for target binding phage. Displayed peptides, geneticallyexpressed on phage coat proteins, are identified by sequencing recoveredphage DNA. A distinct advantage of phage display is that it is anon-biased approach that does not require prior knowledge of thetargeted cell surface receptor. However, selection against a mixedpopulation of differentiated hPS cells is challenging because thecellular heterogeneity limits the abundance of each of the various celltype specific surface targets. Clonal expansion of cells derived fromhPS cell differentiation could provide a more abundant source ofprogenitor cell surface targets for phage selection. Over 140 distinctclonal embryonic progenitor cell lines have been derived from hES cellsusing a combinatorial cell cloning approach (the ACTCellerateInitiative) that resulted in a diverse assortment of clonally pure,scalable cell lines that were selected under a variety of cell cultureand differentiation conditions (West et al. (2008) Regen Med 3: 287).Characterization of these clonal progenitors could result in theidentification of markers associated with progenitor cell types ofspecific organs and tissues allowing for both enrichment of specificprogenitors from a mixed population of cells, as well as the monitoringof the development potential of these progenitor cells both in vivo andin vitro.

The instant invention addresses a variety of needs known in the art,including, but not limited to, the identification of markers associatedwith progenitor cell lines and the enrichment of progenitor cell typesfor use in research and clinical applications.

SUMMARY OF THE INVENTION

In certain embodiments the invention provides a method of enriching apopulation of progenitor cells, wherein the progenitor cell is the invitro progeny of pluripotent stem cell, comprising contacting apopulation of cells comprising the progenitor cell with a peptide thatbinds specifically to the progenitor cell and separating the progenitorcell bound to the peptide from the population of cells thereby enrichingfor a population of progenitor cells.

In other embodiments the invention provides a method of enriching apopulation of mesoderm progenitor cells, wherein the mesoderm progenitorcell is the in vitro progeny of pluripotent stem cell, comprisingcontacting a population of cells comprising the mesoderm progenitor cellwith a peptide that binds specifically to the mesoderm progenitor celland separating the progenitor cell bound to the peptide from thepopulation of cells thereby enriching for a population of mesodermprogenitor cells.

In yet other embodiments the invention provides a method of enriching apopulation of endoderm progenitor cells, wherein the endoderm progenitorcell is the in vitro progeny of pluripotent stem cell, comprisingcontacting a population of cells comprising the endoderm progenitor cellwith a peptide that binds specifically to the endoderm progenitor celland separating the progenitor cell bound to the peptide from thepopulation of cells thereby enriching a population of endodermprogenitor cells thereby enriching for a population of endodermprogenitor cells.

In still other embodiments the invention provides a method of enrichinga population of progenitor cells, wherein the progenitor cells are thein vitro progeny of pluripotent stem cell, comprising contacting apopulation of cells comprising the progenitor cells with a peptidechosen from a peptide comprising an amino acid sequence chosen fromSWTYSYPNQNMD (SEQ ID NO: 1); DWTYSLPGLVEE (SEQ ID NO: 2);NWTWSMPTGNPA(SEQ ID NO: 3); GMTLRVLTN-YTE- (SEQ ID NO: 4); TLHVSENSWTYN(SEQ ID NO: 5); DWLWSFAPNVDT (SEQ ID NO: 6); TLSSQNPYMHKK (SEQ ID NO:7); IDKQMMTSHKAI (SEQ ID NO: 8); QGMETQKLRMLK (SEQ ID NO: 9);GWYWETPLDMFN (SEQ ID NO: 10); GWVIDYDYYPMR (SEQ ID NO: 11); VTAENYQSFSVS(SEQ ID NO: 12); NNKMDDRMMMSIV (SEQ ID NO: 13); STGTDLHSNARI (SEQ ID NO:14); YEFDNLLNRTLW (SEQ ID NO: 15); EWTVNERTMWDL (SEQ ID NO: 16) andseparating the progenitor cell bound to the peptide comprising the aminoacid sequence chosen from SWTYSYPNQNMD (SEQ ID NO: 1); DWTYSLPGLVEE (SEQID NO: 2); NWTWSMPTGNPA(SEQ ID NO: 3); GMTLRVLTN-YTE- (SEQ ID NO: 4);TLHVSENSWTYN (SEQ ID NO: 5); DWLWSFAPNVDT (SEQ ID NO: 6); TLSSQNPYMHKK(SEQ ID NO: 7); IDKQMMTSHKAI (SEQ ID NO: 8); QGMETQKLRMLK (SEQ ID NO:9); GWYWETPLDMFN (SEQ ID NO: 10); GWVIDYDYYPMR (SEQ ID NO: 11);VTAENYQSFSVS (SEQ ID NO: 12); NNKMDDRMMMSIV (SEQ ID NO: 13);STGTDLHSNARI (SEQ ID NO: 14); YEFDNLLNRTLW (SEQ ID NO: 15); EWTVNERTMWDL(SEQ ID NO: 16) from the population of cells thereby enriching for theprogenitor cells. The progenitor cell may be a mesoderm progenitor, oran endoderm progenitor, such as definitive endoderm progenitor forexample.

In further embodiments the invention provides a method of enriching apopulation of cells comprising W10 progenitor cells comprisingcontacting a population of cells comprising the W10 progenitor cell witha peptide that binds to the W10 progenitor cell and separating the W10progenitor cell bound to the peptide from the population of cells.

In further embodiments the invention provides a method of enriching apopulation of progenitor cells expressing one or more of the geneschosen from heart and neural crest derivatives-expressed 2 (HAND2),HOXA4 and HOXB7, wherein the progenitor cell expressing one or more ofthe genes chosen from heart and neural crest derivatives-expressed 2(HAND2), HOXA4 and HOXB7 is the in vitro progeny of pluripotent stemcell, comprising contacting a population of cells comprising theprogenitor cell expressing one or more of the genes chosen from heartand neural crest derivatives-expressed 2 (HAND2), HOXA4 and HOXB7 with apeptide that binds specifically to the progenitor cell expressing one ormore of the genes chosen from heart and neural crestderivatives-expressed 2 (HAND2), HOXA4 and HOXB7 and separating theprogenitor cell bound to the peptide from the population of cellsthereby enriching a population of progenitor cells expressing one ormore of the genes chosen from heart and neural crestderivatives-expressed 2 (HAND2), HOXA4 and HOXB7.

In some embodiments the invention provides a method of enriching apopulation of progenitor cells which expresses one or more genesexpressed by a smooth muscle cell when cultured with TGFβ3, wherein theprogenitor cell expressing one or more genes expressed by a smoothmuscle cell when cultured with TGFβ3 is the in vitro progeny of apluripotent stem cell, comprising contacting a population of cellscomprising the progenitor cell expressing one or more genes expressed bya smooth muscle cell when cultured with TGFβ3 with a peptide that bindsto the progenitor cell expressing one or more genes expressed by asmooth muscle cell when cultured with TGFβ3, and separating theprogenitor cell bound to the peptide from the population of cellsthereby enriching the population of cells expressing one or more genesexpressed by a smooth muscle cell when cultured with TGFβ3.

In yet further embodiments the invention provides a method of enrichinga population of progenitor cells, wherein the progenitor cell is the invitro progeny of pluripotent stem cell, comprising contacting apopulation of cells comprising the progenitor cell with a peptide havingPLEXIN homology that binds specifically to the progenitor cell andseparating the progenitor cell bound to the peptide having PLEXINhomology from the population of cells thereby enriching for thepopulation of progenitor cells. The peptide may be substantiallyhomologous or completely homologous to PLEXIN.

In certain embodiments the invention provides a method separating apluripotent stem cell from a population of cells comprising apluripotent stem cell and a progenitor cell comprising contacting thepopulation of cells comprising a pluripotent stem cells and a progenitorcell with a peptide that binds specifically to a progenitor cell andseparating the progenitor cell bound to the peptide from the populationof cells, thereby separating a pluripotent stem cell from a populationof cells comprising a pluripotent stem cell and a progenitor cell.

In still other embodiments the invention provides a method of separatingan ectoderm progenitor cell from a population of cells comprisingectoderm progenitor cells and either mesoderm progenitor cells, endodermprogenitor cells or both, wherein at least one of the ectodermprogenitor cells, the mesoderm progenitor cells and the endodermprogenitor cells are the in vitro progeny of a pluripotent stem cell,comprising contacting the population of cells comprising an ectodermprogenitor cell and mesoderm progenitor cells, endoderm progenitor cellsor both with a peptide that does not bind to the ectoderm progenitorcell and separating the cells bound to the peptide from the cells thatare not bound to the peptide thereby separating the ectoderm progenitorcells from the population of cells comprising either mesoderm progenitorcells, endoderm progenitor cells or both.

In certain embodiments the invention provides a peptide that bindsspecifically to a progenitor cell, wherein the progenitor cell is the invitro progeny of a pluripotent stem cell.

In some embodiments the invention provides a peptide that bindsspecifically to an endoderm progenitor cell, wherein the progenitor cellis the in vitro progeny of a pluripotent stem cell.

In other embodiments the invention provides a peptide that bindsspecifically to a mesoderm progenitor cell, wherein the mesodermprogenitor cell is the in vitro progeny of a pluripotent stem cell.

In certain embodiments the invention provides a peptide that bindsspecifically to the progenitor cell W10.

In yet other embodiments the invention provides a peptide that bindsspecifically to progenitor cell expressing a protein found in smoothmuscle cell progenitor, wherein the progenitor cell is the in vitroprogeny of a pluripotent stem cell.

In still other embodiments the invention provides a peptide that bindsspecifically to a progenitor cell expressing a protein chosen from heartand neural crest derivatives-expressed 2 (HAND2), HOXA4 and HOXB7,wherein the progenitor cell is the in vitro progeny of a pluripotentstem cell.

In some embodiments the invention provides a peptide that bindsspecifically to progenitor cell expressing at least one gene found inendoderm cells, wherein the progenitor cell is the in vitro progeny of apluripotent stem cell.

In certain embodiments the invention provides a peptide that bindsspecifically to a progenitor cell expressing at least one gene found ina mesoderm cell, wherein the progenitor cell is the in vitro progeny ofa pluripotent stem cell.

In further embodiments the invention provides a peptide that does notbind specifically to an ectoderm progenitor cell.

In other embodiments the invention provides a peptide that bindsspecifically to a progenitor cell, but does not bind to a pluripotentstem cell, wherein the progenitor cell is the in vitro progeny of apluripotent stem cell.

In still further embodiments the invention provides a progenitor cellbinding peptide chosen from the amino acid sequence SWTYSYPNQNMD (SEQ IDNO: 1); DWTYSLPGLVEE (SEQ ID NO: 2); NWTWSMPTGNPA(SEQ ID NO: 3);GMTLRVLTN-YTE- (SEQ ID NO: 4); TLHVSENSWTYN (SEQ ID NO: 5); DWLWSFAPNVDT(SEQ ID NO: 6); TLSSQNPYMHKK (SEQ ID NO: 7); IDKQMMTSHKAI (SEQ ID NO:8); QGMETQKLRMLK (SEQ ID NO: 9); GWYWETPLDMFN (SEQ ID NO: 10);GWVIDYDYYPMR (SEQ ID NO: 11); VTAENYQSFS VS (SEQ ID NO: 12);NNKMDDRMMMSIV (SEQ ID NO: 13); STGTDLHSNARI (SEQ ID NO: 14);YEFDNLLNRTLW (SEQ ID NO: 15); EWTVNERTMWDL (SEQ ID NO: 16).

In still other embodiments the invention provides a progenitor cellbinding peptide wherein the progenitor cell binding peptide comprisesPLEXIN homology and wherein the progenitor cell is the in vitro progenyof a pluripotent stem cell. The peptide may be substantially homologousor completely homologous to PLEXIN.

In other embodiments the invention provides a composition comprising aprogenitor cell and a peptide specifically bound to the progenitor cell,wherein the progenitor cell is the in vitro progeny of a pluripotentstem cell.

In still other embodiments the invention provides a compositioncomprising an endoderm progenitor cell and a peptide bound specificallyto the endoderm progenitor cell, wherein the progenitor cell is the invitro progeny of a pluripotent stem cell.

In further embodiments the invention provides a composition comprising amesoderm progenitor cell and a peptide bound to the mesoderm progenitorcell, wherein the progenitor cell is the in vitro progeny of apluripotent stem cell.

In yet other embodiments the invention provides a composition comprisinga smooth muscle progenitor cell and a peptide bound specifically to thesmooth muscle progenitor cell, wherein the progenitor cell is the invitro progeny of a pluripotent stem cell.

In still other embodiments the invention provides a compositioncomprising the W10 progenitor cell and a peptide specifically bound tothe W10 progenitor cell.

In some embodiments the invention provides a progenitor cell expressinga protein chosen from heart and neural crest derivatives-expressed 2(HAND2), HOXA4 and HOXB7 and a peptide bound specifically to theprogenitor cell expressing a protein chosen from heart and neural crestderivatives-expressed 2 (HAND2), HOXA4 and HOXB7, wherein the progenitorcell is the in vitro progeny of a pluripotent stem cell.

In still further embodiments the invention provides a compositioncomprising a progenitor cell and a peptide bound specifically to theprogenitor cell, wherein the peptide is chosen from a peptide having theamino acid sequence SWTYSYPNQNMD (SEQ ID NO: 1); DWTYSLPGLVEE (SEQ IDNO: 2); NWTWSMPTGNPA(SEQ ID NO: 3); GMTLRVLTN-YTE- (SEQ ID NO: 4);TLHVSENSWTYN (SEQ ID NO: 5); DWLWSFAPNVDT (SEQ ID NO: 6); TLSSQNPYMHKK(SEQ ID NO: 7); IDKQMMTSHKAI (SEQ ID NO: 8); QGMETQKLRMLK (SEQ ID NO:9); GWYWETPLDMFN (SEQ ID NO: 10); GWVIDYDYYPMR (SEQ ID NO: 11);VTAENYQSFSVS (SEQ ID NO: 12); NNKMDDRMMMSIV (SEQ ID NO: 13);STGTDLHSNARI (SEQ ID NO: 14); YEFDNLLNRTLW (SEQ ID NO: 15); EWTVNERTMWDL(SEQ ID NO: 16), wherein the progenitor cell is the in vitro progeny ofa pluripotent stem cell.

In yet other embodiments the invention provides a composition comprisinga progenitor cell and a peptide bound specifically to the progenitorcell, wherein the peptide bound specifically to the progenitor cellcomprises PLEXIN homology, wherein the progenitor cell is the in vitroprogeny of a pluripotent stem cell. The peptide may be substantiallyhomologous or completely homologous to PLEXIN.

In further embodiments the invention provides a method of detecting aprogenitor cell comprising contacting the progenitor cell with a peptidethat binds the progenitor cell specifically, wherein the peptidecomprises a detectable substance, thereby detecting the progenitor cell.The progenitor cell may be detected in vitro or in vivo.

In still other embodiments the invention provides a method of detectinga mesoderm progenitor cell comprising contacting the mesoderm progenitorcell with a peptide that binds specifically to the mesoderm progenitorcell, wherein the peptide comprises a detectable substance, therebydetecting the progenitor cell. The progenitor cell may be detected invitro or in vivo.

In other embodiments the invention provides a method of detecting anendoderm progenitor cell comprising contacting the endoderm progenitorcell with a peptide that binds specifically to the endoderm progenitorcell, wherein the peptide comprises a detectable substance, therebydetecting the progenitor cell. The progenitor cell may be detected invitro or in vivo.

In still other embodiments the invention provides a method of detectinga smooth muscle progenitor cell comprising contacting the smooth muscleprogenitor cell with a peptide that binds specifically to the smoothmuscle progenitor cell, wherein the peptide comprises a detectablesubstance, thereby detecting the progenitor cell. The progenitor cellmay be detected in vitro or in vivo.

In yet other embodiments the invention provides a method of detecting aW10 progenitor cell comprising contacting the W10 progenitor cell with apeptide that binds specifically to the W10 progenitor cell, wherein thepeptide comprises a detectable substance, thereby detecting theprogenitor cell. The progenitor cell may be detected in vitro or invivo.

In further embodiments the invention provides a method of detecting aprogenitor cell comprising contacting the progenitor cell with a peptidethat binds the progenitor cell specifically, wherein the peptide ischosen from a peptide having the amino acid sequence SWTYSYPNQNMD (SEQID NO: 1); DWTYSLPGLVEE (SEQ ID NO: 2); NWTWSMPTGNPA(SEQ ID NO: 3);GMTLRVLTN-YTE- (SEQ ID NO: 4); TLHVSENSWTYN (SEQ ID NO: 5); DWLWSFAPNVDT(SEQ ID NO: 6); TLSSQNPYMHKK (SEQ ID NO: 7); IDKQMMTSHKAI (SEQ ID NO:8); QGMETQKLRMLK (SEQ ID NO: 9); GWYWETPLDMFN (SEQ ID NO: 10);GWVIDYDYYPMR (SEQ ID NO: 11); VTAENYQSFSVS (SEQ ID NO: 12);NNKMDDRMMMSIV (SEQ ID NO: 13); STGTDLHSNARI (SEQ ID NO: 14);YEFDNLLNRTLW (SEQ ID NO: 15); EWTVNERTMWDL (SEQ ID NO: 16) and whereinthe peptide comprises a detectable substance, thereby detecting theprogenitor cell. The progenitor cell may be detected in vitro or invivo.

In further embodiments the invention provides a method of detecting aprogenitor cell expressing a protein chosen from heart and neural crestderivatives-expressed 2 (HAND2), HOXA4 and HOXB7 comprising contactingthe progenitor cell expressing a protein chosen from heart and neuralcrest derivatives-expressed 2 (HAND2), HOXA4 and HOXB7 with a peptidethat binds specifically to the progenitor cell expressing a proteinchosen from heart and neural crest derivatives-expressed 2 (HAND2),HOXA4 and HOXB7, wherein the peptide comprises a detectable substance,thereby detecting the progenitor cell. The progenitor cell may bedetected in vitro or in vivo.

In yet other embodiments the invention provides a method of detecting aprogenitor cell comprising contacting the progenitor cell with a peptidehaving PLEXIN homology and that binds specifically to the progenitorcell, wherein the peptide comprises a detectable substance, therebydetecting the progenitor cell. The progenitor cell may be detected invitro or in vivo. The peptide may be substantially homologous orcompletely homologous to PLEXIN

In certain embodiments the invention provides a method of monitoring thedifferentiation of a progenitor cell comprising 1) contacting theprogenitor cell with a peptide that binds to the progenitor cell,wherein the peptide that binds to the progenitor cell comprises adetectable substance and 2) monitoring the progenitor cell bound to thepeptide over time thereby monitoring the differentiation of a progenitorcell. The cell may be monitored in vivo or in vitro.

In further embodiments the invention provides a method of monitoring thedifferentiation of an endoderm progenitor cell comprising 1) contactingthe endoderm progenitor cell with a peptide that binds specifically tothe endoderm progenitor cell, wherein the peptide that bindsspecifically to the endoderm progenitor cell comprises a detectablesubstance and 2) monitoring the progenitor cell bound to the peptideover time thereby monitoring the differentiation of an endodermprogenitor cell. The cell may be monitored in vivo or in vitro.

In still further embodiments the invention provides a method ofmonitoring the differentiation of an mesoderm progenitor cellcomprising 1) contacting the mesoderm progenitor cell with a peptidethat binds specifically to the mesoderm progenitor cell, wherein thepeptide that binds specifically to the mesoderm progenitor cellcomprises a detectable substance and 2) monitoring the progenitor cellbound to the peptide over time thereby monitoring the differentiation ofa mesoderm progenitor cell. The cell may be monitored in vivo or invitro.

In still other embodiments the invention provides a method of monitoringthe differentiation of a smooth muscle progenitor cell comprising 1)contacting the smooth muscle progenitor cell with a peptide that bindsspecifically to the smooth muscle progenitor cell, wherein the peptidethat binds specifically to the smooth muscle progenitor cell comprises adetectable substance and 2) monitoring the progenitor cell bound to thepeptide over time thereby monitoring the differentiation of a progenitorcell. The cell may be monitored in vivo or in vitro.

In further embodiments the invention provides a method of monitoring thedifferentiation of a W10 cell comprising contacting the W10 cell with apeptide that binds specifically to the W10 cell, wherein the peptidethat binds specifically to the W10 cell comprises a detectable substanceand monitoring the progenitor cell bound to the peptide over timethereby monitoring the differentiation of a progenitor cell. The cellmay be monitored in vivo or in vitro.

In certain embodiments the invention provides a method of monitoring thedifferentiation of a progenitor cell expressing a protein chosen fromheart and neural crest derivatives-expressed 2 (HAND2), HOXA4 and HOXB7comprising 1) contacting the progenitor cell expressing a protein chosenfrom heart and neural crest derivatives-expressed 2 (HAND2), HOXA4 andHOXB7 with a peptide that binds to the progenitor cell expressing aprotein chosen from heart and neural crest derivatives-expressed 2(HAND2), HOXA4 and HOXB7, wherein the peptide that binds to theprogenitor cell comprises a detectable substance and monitoring theprogenitor cell bound to the peptide over time thereby monitoring thedifferentiation of a progenitor cell. The cell may be monitored in vivoor in vitro.

In some embodiments the invention provides a method of monitoring thedifferentiation of a progenitor cell comprising 1) contacting theprogenitor cell with a peptide that binds to the progenitor cell,wherein the peptide is chosen from a peptide having the amino acidsequence SWTYSYPNQNMD (SEQ ID NO: 1); DWTYSLPGLVEE (SEQ ID NO: 2);NWTWSMPTGNPA(SEQ ID NO: 3); GMTLRVLTN-YTE- (SEQ ID NO: 4); TLHVSENSWTYN(SEQ ID NO: 5); DWLWSFAPNVDT (SEQ ID NO: 6); TLSSQNPYMHKK (SEQ ID NO:7); IDKQMMTSHKAI (SEQ ID NO: 8); QGMETQKLRMLK (SEQ ID NO: 9);GWYWETPLDMFN (SEQ ID NO: 10); GWVIDYDYYPMR (SEQ ID NO: 11); VTAENYQSFSVS(SEQ ID NO: 12); NNKMDDRMMMSIV (SEQ ID NO: 13); STGTDLHSNARI (SEQ ID NO:14); YEFDNLLNRTLW (SEQ ID NO: 15); EWTVNERTMWDL (SEQ ID NO: 16) andwherein the peptide that binds to the progenitor cell comprises adetectable substance and 2) monitoring the progenitor cell bound to thepeptide over time thereby monitoring the differentiation of a progenitorcell. The cell may be monitored in vivo or in vitro.

In still other embodiments the invention provides a kit comprising oneor more peptides that bind specifically to a progenitor cell, whereinthe progenitor cell is the in vitro progeny of a pluripotent stem cell.

DESCRIPTION OF DRAWINGS

For a fuller understanding of the nature and advantages of the presentinvention, reference should be had to the following detailed descriptiontaken in connection with the accompanying drawings, in which:

FIG. 1A and FIG. 1B. W10 is a progenitor cell line capable of smoothmuscle differentiation. FIG. 1A: Undifferentiated (day 0) anddifferentiated W10 micromass (MM) cultures in the presence of 10 ng/mlTGFβ3 (day 14). Cells were stained with anti-MYH11 antibody and DAPI.FIG. 1B: W10 cells express smooth muscle marker, MYH11, but notcartilage marker COL2A1 upon 14 day MM differentiation (as in A). Meanexpression of the MYH11 and COL2A1 by Illumina microarray of day 0undifferentiated control and day 14 differentiated MM cultures ofcoronary artery smooth muscle cell (CASMC), W10, and the chondrogeniccell line, 4D20.8. Values are from duplicate (CASMC) and triplicate (W10and 4D20.8) experiments.

FIG. 2A through FIG. 2D. Selection of a peptide phage display libraryagainst W10 progenitor cells. FIG. 2A: Peptide phages that bind to W10progenitor cell line were enriched by 3 rounds of biopanning PhD-12phage display peptide library (2×10¹¹ pfu, for round 1) or amplifiedrecovered phage (2×10¹⁰ pfu, for rounds 2 and 3) were first adsorbedagainst human adult dermal fibroblasts cells and then incubated withadherent W10 cells. The phage were recovered from the cell lysate andsample phage clones were sequenced. The enriched library was amplifiedfor further rounds of selection. FIG. 2B: The percentage of input phagerecovered increased with each round of selection. The percentage ofinput phage recovered was determined by titration of plaque formingunits (pfu) in the cell lysate relative to the input pfu used for eachpanning round. FIG. 2C: Frequency and multiple sequence alignment ofpeptides identified as candidate peptide phage in rounds 2 and 3 ofpanning generated by CLUSTAL W (2.10). FIG. 2D: Phylogram based on FIG.2C denoting peptide similarities.

FIG. 3A and FIG. 3B. Binding of peptide display phages to W10 embryonicprogenitor cell line. FIG. 3A: Immunofluorescent detection of boundphages. Cells were incubated with 2×10¹⁰ phage particles for 2 h at 37°C.; unbound phages were removed by washing and cells were fixed andpermeabilized. Bound phages were detected by immunocytochemistry usingrabbit anti-phage antibody and Alexa568-conjugated goat anti-rabbitantibody. Cell nuclei were stained using DAPI. FIG. 3B: Quantitation ofpeptide phage cell binding. 2×10¹⁰ pfu of each candidate or controls(RGD, Gly12 and empty phage M13KE) phages were assessed for binding on1×10⁵ W10 progenitor cells for 2 h at 37° C. Cell associated phages wererecovered from cell lysates and quantified by titration. Protein in celllysates was measured by microBCA assay. The relative binding factor (BF)is calculated as peptide phage recovery (percentage of input) relativeto M13KE control phage recovery (percentage of input). Values are fromtriplicate experiments and shown as mean +/−standard error. BFs for the4 W10 peptide phage were statistically significant from the controlM13KE phage (ANOVA with Dunnett's multiple comparison tests; p values:*: 0.05 and **: 0.01). BFs for RGD and Gly12 were not statisticallysignificant.

FIG. 4A and FIG. 4B. Phage binding competition with free peptide.Competition of the peptide phage with free peptide was measured usingFIG. 4A: Immunofluorescent detection of bound peptide phages. Chemicallysynthesized peptides were added to compete with binding of peptidephages to W10 progenitor cells. Cells were pre-incubated with differentpeptides at 100 μM or without peptide for 30 min at 4° C., followed bypeptide phages (2×10¹⁰ pfu) for an additional 1 h at 4° C. Afterwashing, the bound peptide phages were detected by immunofluorescence.Peptide sequences are: W10 R2 11 biotin: GWVIDYDYYPMRGGGK(biotin) (SEQID NO: 17); FITC W10 R2 11: FITC GWVIDYDYYPMRGGG (SEQ ID NO: 18) andFITC-unrelated: FITC NHVHRMHATPAY (SEQ ID NO: 19). FIG. 4B: Percentageof input phage recovered from cell lysate. Cells were pre-incubated withpeptides at 5 μM or 5 nM, or without peptide for 30 min at 4° C.,followed by peptide phages (2×10¹⁰ pfu) for an additional 1 h at 4° C.After washing, the recovered phage was quantified by titration. Thecompetition is shown as percentage of no-peptide control. Values arefrom triplicate experiments shown as mean +/−standard error. Competitionby the corresponding free peptide was statistically significant at 5 nMand 5 μM with the exception of W10-R2-21 (only significant at 5 μM).Competition by scrambled or unrelated peptide was not statisticallysignificant. (ANOVA with Dunnett's multiple comparison tests; p values:*: 0.05. **: 0.01 and ***: 0.001). Peptide sequences are: peptide:X12GGGK(biotin)(SEQ ID NO: 74); unrelated: biotin NHVHRMHATPAY(SEQ IDNO: 19); W10 R2 11 scrambled: DYWDVGPIYRMYGGGG (SEQ ID NO: 75); W10 R221 scrambled: LGTMDWFWPYNEGGGG (SEQ ID NO: 20); W10-R3-18-scrambled:VSDPFDNLWTAWGGGK (SEQ ID NO: 21).

FIG. 5A through FIG. 5C. Labeling of embryonic progenitor cell lineusing peptide targeted Qdot605. FIG. 5A: Cell targeting by fluorescentQdots. Qdot605 ITK SA were complexed with an excess of chemicallysynthesized C-terminal biotinylated peptide; unbound peptide was removedby dialysis. W10 progenitor cells were incubated for 16 h at 37° C. with5 nM of Qdot complexes, washed and imaged using a fluorescencemicroscope. FIG. 5B: Competition with free peptide or peptide-targetedQdots. Cells were pre-incubated with 5 nM peptide, peptide targetedQdots, or untargeted Qdots, for 30 min at 4° C., followed by addition ofpeptide phage (2×10¹⁰ pfu) for an additional 1 h at 4° C. After washing,the recovered phage was quantified by titration. The competition isshown as percentage of no-peptide control. Values are from triplicateexperiments and shown as mean +/−standard error. Competition bycorresponding free peptide or peptide-Qdot complex at 5 nM wasstatistically significant. Competition by uncoupled Qdots was notstatistically significant (ANOVA with Dunnett's multiple comparisontests; p values: *: 0.05. **: 0.01 and ***: 0.001). FIG. 5C: Flowcytometry analysis. Cells were labeled as in (A), dissociated from thetissue culture plate using TrypLE, resuspended in PBS and analyzed inLSRFortessa flow cytometer. 10,000 events were recorded for each sample;cells were excited using the 405 nm laser and fluorescence emission wasdetected with the 605/12 bandpass filter. Cells labeled with W10 R3 18peptide-Qdot complexes (green) showed higher mean fluorescent intensitythan cells labeled with untargeted Qdots (red) or unlabeled W10 cells(blue).

FIG. 6. Selectivity of Qdot peptide complexes. Embryonic progenitor celllines were labeled with Qdot complexes in their corresponding growthmedia and analyzed by flow cytometry as in (FIG. 5C). Percentage oflabeled cells was calculated by setting up gates (allowing up to 1%)using the progenitor cell line labeled with untargeted Qdots andunlabeled cells. 10,000 events were recorded for each sample. Values arefrom triplicate experiments and shown as mean +/−standard error.

FIG. 7A and FIG. 7B. Labeling of a differentiating pluripotent stemcells using peptide targeted Qdots. FIG. 7A: Selective peptide targetingof human embryonic stem cells (H9) that were differentiated into thethree germ layers (see methods for protocols) for 6-8 days compared toundifferentiated control. Differentiated cells were incubated for 16 hat 37° C. with 5 nM of Qdot complexes, washed and imaged using afluorescence microscope. Representative bright-field and fluorescentsignal of W10 peptide Qdot complexes (red) are shown. FIG. 7B: Cellswere stained with the following differentiation markers to verify thegerm layer commitment: nestin for ectoderm, α-actinin for mesoderm,SOX17 for endoderm conditions and OCT3/4 for undifferentiated cells.

FIG. 8A through FIG. 8E. Selectivity of Qdot peptide complexes. FIG. 8A:Fluorescence microscopy images of confluent embryonic progenitor celllines 4D20.8 and 7PEND24 labeled with W10-peptide Qdot complexes,showing only signal from Qdot655 channel only and corresponding overlaphistograms of flow cytometric quantification of the same labeled celllines. FIG. 8B: Fluorescence microscopy images of confluent embryonicprogenitor cell lines E15 and J16 labeled with W10-peptide Qdotcomplexes, showing only signal from Qdot655 channel only andcorresponding overlap histograms of flow cytometric quantification ofthe same labeled cell lines. FIG. 8C: Fluorescence microscopy images ofconfluent embryonic progenitor cell lines J8 and MW1 labeled withW10-peptide Qdot complexes, showing only signal from Qdot655 channelonly and corresponding overlap histograms of flow cytometricquantification of the same labeled cell lines. FIG. 8D: Fluorescencemicroscopy images of confluent embryonic progenitor cell lines SM30 andU31 labeled with W10-peptide Qdot complexes, showing only signal fromQdot655 channel only and corresponding overlap histograms of flowcytometric quantification of the same labeled cell lines. FIG. 8E:Fluorescence microscopy images of confluent embryonic progenitor celllines W10 and Z11 labeled with W10-peptide Qdot complexes, showing onlysignal from Qdot655 channel only and corresponding overlap histograms offlow cytometric quantification of the same labeled cell lines. W10peptide Qdot complex is shown in red while control samples of uncoupledQdots and unstained cells are shown in black and grey, respectively.Results are representative of three independent experiments.

FIG. 9A through FIG. 9C. Differentiation of W10 cell line. Geneexpression analysis of cultured coronary artery smooth muscle, W10, and4D20.8 cells in the undifferentiated state and micromass (MM)differentiation conditions. FIG. 9A and FIG. 9B: Comparative microarrayrelative fluorescence units (RFU) values for coronary artery smoothmuscle cells, W10, and 4D20.8 in control conditions of five-dayquiescence and 14 days of micromass culture. FIG. 9C: Values fromselected genes are compiled from data in FIG. 9A and FIG. 9B, and thecorresponding graphs showed the upregulation of smooth muscle heavychain 11 (MYH11), calponin 1 (CNN1), myosin light chain kinase (MYLK),and smooth muscle actin (ACTA2) in W10 and CASMC cells but not in 4D20.8cells under myodifferentiation conditions.

FIG. 10A through FIG. 10C. Analysis of binding W10 peptide phagesequences. FIG. 10A: Best score hit for homologous protein sequenceswere identified in the Homo sapiens RefSeq protein database using Blastp(PSI-Blast, position-specific iterated BLAST with word size of 3 andBlosum62 matrix, available online at: blast.ncbi.nlm nih gov). FIG. 10B:Sequence homology of the W10 binding peptides with plexins andsemaphorin. Identical amino acids are in bold, highly similar are grey.FIG. 10C lists the SEQ ID NOS of the sequences provided in FIG. 10A andFIG. 10B.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present disclosure, the suitable methods, devices, and materialsare now described. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Previous studies have shown that phage display is useful for identifyingpeptides that target undifferentiated (Lu et al. (2010) PLoS One 5:e12075; Zhao et al. (2010) J Biomol Screen 15: 687; Zhao et al. (2010)Peptides 31: 2027), differentiated cells (Zhao et al. (2010) J BiomolScreen 15: 687) or cancer cell lines (Rasmussen et al. (2002) CancerGene Ther 9: 606; Spear et al. (2001) Cancer Gene Ther 8: 506). Wedescribe here a novel phage display strategy that uses selection againsta clonally pure pluripotent stem cell derivative to identify peptidesthat selectively target early human pluripotent stem (hPS) cell derivedprogenitor cell populations. The peptides developed here clearly bind toone or more developmentally regulated surface markers that are absent onundifferentiated pluripotent stem cells but are detected predominantlyon definitive endodermal progenitors derived from 6-8 daydifferentiating hPS cells. Peptide targeting to definitive endoderm wasunexpected given that the peptides were selected on the W10 cell linewhich expresses smooth muscle and other mesodermal markers (West et al.(2008) Regen Med 3: 287). However, the targets for the cell bindingpeptides although restricted may be present on more than one progenitorcell type. Analysis of 10 ACTCellerate cell lines revealed highlyprevalent peptide binding to multiple distinct progenitor cell lines.

Cellular heterogeneity in hPS derived cell populations is a majorbottleneck for the successful development of hPS derived cells fortransplantation. Contamination of differentiated cells with residualpluripotent cells is of particular concern for safety because of theirability to form teratomas in vivo (Blum, Benvenisty (2009) Cell Cycle 8:3822). Cell purity is also important for consistency of non-clinicalapplications such as disease modeling, drug screening and drug safetytesting. We have begun to address this issue by developing targetingpeptides that can identify subsets of progenitor cell types for use incell enrichment and cell exclusion procedures. The advantage of suchcell enrichment steps is clearly demonstrated by over a decade of theclinical application of cell surface targeted enrichment ofhematopoietic stem cells for stem cell transplants as a cancer treatment(Grutzkau, Radbruch (2010) Cytometry A 77: 643). We isolated humanprogenitor stem cell targeting peptides that recognize certain hPSderived progenitor stem cell lines as well as hPS derived earlydefinitive endoderm. The endodermal progenitor targeting peptides mightbe useful for enriching or excluding endodermal progenitors duringdirected differentiation. The peptide targeted Qdots could also be usedto rapidly assess hPS cell differentiation capacity and to screen forreagents that direct differentiation toward definitive endoderm.Identification of additional progenitor stem cell targeting peptidesusing the approach described here may make it possible to improverecovery of clinically relevant progenitor cell types. This would beparticularly useful for deriving patient-specific progenitors from thepatient's own reprogrammed iPS cells. For example, a recent preclinicalstudy of one of the ACTCellerate clonal cell lines, 4D20.8, hasdemonstrated the ability of this cell line to differentiate tochondrocytes capable of cartilage repair in a rat knee model (Sternberget al. (2012) Regen Med 7: 481). It may be feasible to use the phagedisplay approach described here to isolate stem cell targeting peptidesthat would facilitate retrieval of the equivalent cells from patientderived iPS cells to provide a source of genetically matched stem cellsfor cell replacement therapy.

In certain embodiments the invention provides peptides that bindspecifically to progenitor cells that have been differentiated in vitro,and thus are the in vitro progeny of pluripotent stem cells. Thepeptides may be used to enrich for, or deplete the target progenitorcell to which it binds. The peptides may be used to visualize and/ormonitor the progenitor cell as it differentiates further down a specificdevelopmental pathway. The peptides may be used to identify and orcharacterize progenitor cell lines. For example the peptides can be usedto identify one or more proteins expressed on the surface of aprogenitor cell. The peptides may be used to block other agents, such asproteins, small molecules, drugs, and the like from binding to the cellsurface of the progenitor cell.

Definitions

As used herein, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, reference to a “therapeutic” is a reference to one or moretherapeutics and equivalents thereof known to those skilled in the art,and so forth.

As used herein, the term “about” means plus or minus 5% of the numericalvalue of the number with which it is being used. Therefore, about 50%means in the range of 45% to 55%.

The term “animal,” “patient” or “subject” as used herein includes, butis not limited to, humans, non-human primates and non-human vertebratessuch as wild, domestic and farm animals including any mammal, such ascats, dogs, cows, sheep, pigs, horses, rabbits, rodents such as mice andrats. In some embodiments, the term “subject,” “patient” or “animal”refers to a male. In some embodiments, the term “subject,” “patient” or“animal” refers to a female.

The term “antibody”, as used herein, means an immunoglobulin or a partthereof, and encompasses any polypeptide comprising an antigen-bindingsite regardless of the source, method of production, or othercharacteristics. The term includes for example, polyclonal, monoclonal,monospecific, polyspecific, humanized, single-chain, chimeric,synthetic, recombinant, hybrid, mutated, and CDR-grafted antibodies. Apart of an antibody can include any fragment which can bind antigen, forexample, an Fab, F (ab′)_(2,) Fv, scFv.

The term “ectoderm” as used herein, refers to the most exterior (ordistal) layer of the developing embryo. The ectoderm differentiates toform the nervous system (spine, peripheral nerves and brain), toothenamel and the epidermis (the outer part of integument). It also formsthe lining of mouth, anus, nostrils, sweat glands, hair and nails. Invertebrates, the ectoderm has three parts: external ectoderm (also knownas surface ectoderm), the neural crest, and neural tube. The latter twoare known as neuroectoderm.

The term “endoderm,” as used herein, refers to the innermost germ layerof the early embryo. It gives rise to the entire alimentary canal exceptpart of the mouth, pharynx and the terminal part of the rectum (whichare lined by involutions of the ectoderm), the lining cells of all theglands which open into the digestive tube, including those of the liverand pancreas; the trachea, bronchi, and alveoli of the lungs; the liningof the follicles of the thyroid gland and thymus; the epithelium of theauditory tube and tympanic cavity; the urinary bladder and part of theurethra.

The term “gene expression result” refers to a qualitative and/orquantitative result regarding the expression of a gene or gene product.Any method known in the art may be used to quantitate a gene expressionresult. The gene expression result can be an amount or copy number ofthe gene, the RNA encoded by the gene, the mRNA encoded by the gene, theprotein product encoded by the gene, or any combination thereof. Thegene expression result can also be normalized or compared to a standard.The gene expression result can be used, for example, to determine if agene is expressed, overexpressed, or differentially expressed in two ormore samples by comparing the gene expression results from 2 or moresamples or one or more samples with a standard or a control.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword “identity” may substitute for the word “homology.” A partiallycomplementary nucleic acid sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as “substantially homologous.” The inhibition of hybridization of thecompletely complementary nucleic acid sequence to the target sequencemay be examined using a hybridization assay (Southern or northern blot,solution hybridization, and the like) under conditions of reducedstringency. A substantially homologous sequence or hybridization probewill compete for and inhibit the binding of a completely homologoussequence to the target sequence under conditions of reduced stringency.This is not to say that conditions of reduced stringency are such thatnon-specific binding is permitted, as reduced stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., a selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% homology oridentity). In the absence of non-specific binding, the substantiallyhomologous sequence or probe will not hybridize to the secondnon-complementary target sequence.

As used herein, the term “hybridization” or “hybridizing” refers tohydrogen bonding, which may be Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding between complementary nucleoside ornucleotide bases. For example, adenine and thymine are complementarynucleobases which pair through the formation of hydrogen bonds.“Complementary,” as used herein in reference to nucleic acid moleculesrefers to the capacity for precise pairing between two nucleotides. Forexample, if a nucleotide at a certain position of an oligonucleotide iscapable of hydrogen bonding with a nucleotide at the same position of aDNA or RNA molecule, then the oligonucleotide and the DNA or RNA areconsidered to be complementary to each other at that position. Theoligonucleotide and the DNA or RNA are complementary to each other whena sufficient number of corresponding positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity or precise pairingsuch that stable and specific binding occurs between the oligonucleotideand the DNA or RNA target. It is understood in the art that a nucleicacid sequence need not be 100% complementary to that of its targetnucleic acid to be specifically hybridizable. A nucleic acid compound isspecifically hybridizable when there is binding of the molecule to thetarget, and there is a sufficient degree of complementarity to avoidnon-specific binding of the molecule to non-target sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

The term “induced pluripotent stem cell,” (iPS) as used herein, refersto a pluripotent cell obtained by manipulating (in vitro) anon-pluripotent cell, such as a somatic cell, so that it reverts back toa pluripotent state similar to the pluripotent state seen in embryonicstem cells.

The term “label” and/or “detectible substance” refers to a compositioncapable of producing a detectable signal indicative of the presence ofthe target polynucleotide or a polypeptide or protein in an assaysample. Suitable labels include radioisotopes, nucleotide chromophores,enzymes, substrates, fluorescent molecules, chemiluminescent moieties,magnetic particles, bioluminescent moieties, and the like. As such, alabel is any composition detectable by a device or method, such as, butnot limited to, a spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, chemical detection device or anyother appropriate device. In some embodiments, the label may bedetectable visually without the aid of a device. The term “label” or“detectable substance” is used to refer to any chemical group or moietyhaving a detectable physical property or any compound capable of causinga chemical group or moiety to exhibit a detectable physical property,such as an enzyme that catalyzes conversion of a substrate into adetectable product. The term “label” or “detectable substance” alsoencompasses compounds that inhibit the expression of a particularphysical property. The label may also be a compound that is a member ofa binding pair, the other member of which bears a detectable physicalproperty.

The term “multipotent cell,” as used herein, refers to a cell that candifferentiate into a plurality of cell types, but which cannotdifferentiate into one or more cells found in each of the three germlayers: endoderm, ectoderm, mesoderm. It is generally moredevelopmentally advanced or mature compared to a pluripotent stem cell.

The term “mesoderm” as used herein, refers to the germ layer that formsmany muscles, the heart, the circulatory and excretory systems, and thedermis, skeleton, and other supportive and connective tissue. It alsogives rise to the notochord, a supporting structure between the neuralcanal and the primitive gut. In many animals, including vertebrates, themesoderm surrounds a cavity known as the coelom, the space that containsthe viscera.

The use of “nucleic acid,” “polynucleotide” or “oligonucleotide” orequivalents herein means at least two nucleotides covalently linkedtogether. In some embodiments, an oligonucleotide is an oligomer of 6,8, 10, 12, 20, 30 or up to 100 nucleotides. In some embodiments, anoligonucleotide is an oligomer of at least 6, 8, 10, 12, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides. A“polynucleotide” or “oligonucleotide” may comprise DNA, RNA, PNA or apolymer of nucleotides linked by phosphodiester and/or any alternatebonds.

As used herein, the term “optional” or “optionally” refers toembodiments where the subsequently described structure, event orcircumstance may or may not occur, and that the description includesinstances where the event occurs and instances where it does not.

The term “passage” and “passaged,” as used herein, refers to splitting agrowing replicating cell culture that has reached a desired cell density(e.g. confluency or 80% confluency for an adherent cell line) into afractional a fractional component such as ½, ⅓ or the like, in order toallow the cells to continue to replicate.

The term “peptide” as used herein, refers to a series of amino acidslinked by peptide bonds. In some instances, a peptide may comprise aportion of a full length protein.

The phrases “percent homology,” “% homology,” “percent identity,” or “%identity” refer to the percentage of sequence similarity found in acomparison of two or more amino acid or nucleic acid sequences. Percentidentity can be determined electronically, e.g., by using the MEGALIGNprogram (LASERGENE software package, DNASTAR). The MEGALIGN program cancreate alignments between two or more sequences according to differentmethods, e.g., the Clustal Method. (Higgins, D. G. and P. M. Sharp(1988) Gene 73:237-244.) The Clustal algorithm groups sequences intoclusters by examining the distances between all pairs. The clusters arealigned pairwise and then in groups. The percentage similarity betweentwo amino acid sequences, e.g., sequence A and sequence B, is calculatedby dividing the length of sequence A, minus the number of gap residuesin sequence A, minus the number of gap residues in sequence B, into thesum of the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no homology between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be calculated by theClustal Method, or by other methods known in the art, such as the JotunHein Method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)Identity between sequences can also be determined by other methods knownin the art, e.g., by varying hybridization conditions.

“Pluripotent stem cell,” as used herein, refers to a cell that has theability, when cultured under the appropriate conditions to differentiateinto a least one cell type from the three embryonic germ layers:ectoderm, endoderm and mesoderm. Examples of pluripotent stem cells,include, but are not limited to, embryonic stem cells, such asestablished embryonic stem cell lines, and induced pluripotent stemcells.

“Progenitor cells,” as used herein, refers to a cell that is no longer apluripotent stem cell in that it has differentiated beyond thepluripotent state, but retains the ability to differentiate further, forexample, into a cell type that expresses at least one gene found in anadult organism, such as a mammal. Examples of progenitor cells includethe W10 cell line, the 4D20.8 cell line, the SM30 cell line, the 7PEND24cell line, and the E15 cell line.

“Progeny of a pluripotent stem cell,” as used herein, refers to one ormore daughter cells obtained from a parental pluripotent stem cell.Included within this definition are instances where the progeny is adifferentiated cell that is no longer a pluripotent stem cell. Examplesof progeny of a pluripotent stem cell may include progenitor cells whichare no longer pluripotent, but are still multipotent, as well as cellsthat have fully differentiated into a mature phenotype. Non-pluripotentprogeny of pluripotent stem cells are sometimes referred to herein as“differentiated progeny of pluripotent stem cells.” The progeny of apluripotent stem cell may be obtained in vitro, i.e. by growing thecells under suitable culture conditions, or in vivo, i.e. bytransplanting the cells into a subject.

“Recombinant protein,” as used herein, means a protein made usingrecombinant techniques, for example, but not limited to, through theexpression of a recombinant nucleic acid as depicted infra. Arecombinant protein may be distinguished from naturally occurringprotein by at least one or more characteristics. For example, theprotein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedprotein is unaccompanied by at least some of the material with which itis normally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesabout 50-75%, about 80%, or about 90%. In some embodiments, asubstantially pure protein comprises about 80-99%, 85-99%, 90-99%,95-99%, or 97-99% by weight of the total protein. A recombinant proteincan also include the production of a protein from one organism (e.g.human) in a different organism (e.g. yeast, E. coli, or the like) orhost cell. Alternatively, the protein may be made at a significantlyhigher concentration than is normally seen, through the use of aninducible promoter or high expression promoter, such that the protein ismade at increased concentration levels. Alternatively, the protein maybe in a form not normally found in nature, as in the addition of anepitope tag or amino acid substitutions, insertions and deletions, asdiscussed herein.

As used herein, the term “specifically binds” or “specifically binding”means binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule, which generally is a molecule of similar structure that doesnot have binding activity. For example, specific binding is indicated ifthe molecule has measurably higher affinity for cells expressing aprotein than for cells that do not express the same protein. Specificityof binding can be determined, for example, by competitive inhibition ofa known binding molecule.

The term “specifically binds” “specifically binding,” as used herein,includes both low and high affinity specific binding. Specific bindingcan be exhibited, for example, by a low affinity homing molecule havinga Kd of at least about 10 ^(−4 M). Specific binding also can beexhibited by a high affinity molecule, for example, a molecule having aKd of at least about 10^(−5 M). Such a molecule can have, for example, aKd of at least about 10^(−6 M), at least about 10^(−7 M), at least about10^(−8 M), at least about 10^(−9 M), at least about 10^(−10 M) or canhave a Kd of at least about 10^(−11 M) or 10^(−12 M) or smaller.Nonspecific binding may be characterized by a KD of 10 ⁻³ or larger.Both low and high affinity binding molecules are useful and areencompassed by the invention. Low affinity homing molecules may beuseful in targeting, for example, multivalent conjugates. High affinitybinding molecules may be useful in targeting, for example, multivalentand univalent conjugates

The terms “treat,” “treated,” or “treating” as used herein can refer toboth therapeutic treatment or prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) an undesiredphysiological condition, symptom, disorder or disease, or to obtainbeneficial or desired clinical results. In some embodiments, the termmay refer to both treating and preventing. For the purposes of thisdisclosure, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms; diminishment of the extent of thecondition, disorder or disease; stabilization (i.e., not worsening) ofthe state of the condition, disorder or disease; delay in onset orslowing of the progression of the condition, disorder or disease;amelioration of the condition, disorder or disease state; and remission(whether partial or total), whether detectable or undetectable, orenhancement or improvement of the condition, disorder or disease.Treatment includes eliciting a clinically significant response withoutexcessive levels of side effects. Treatment also includes prolongingsurvival as compared to expected survival if not receiving treatment.

The term “tissue” refers to any aggregation of similarly specializedcells that are united in the performance of a particular function.

Methods of Enriching for Progenitor Cells

In certain embodiments the invention provides a method of enriching apopulation of progenitor cells comprising contacting the progenitor cellwith a peptide that specifically binds to the progenitor cell andseparating the progenitor cells bound to the peptide from those cellsthat are not bound to the peptide. Peptides that are suitable for use inthe method include any peptide described infra. In some embodiments thepeptide may comprise, for example, any of the amino acid sequencesdisclosed infra. Progenitor cells that are suitable for use in themethod are any progenitor cell described infra.

In other embodiments the invention provides a method of eliminatingunwanted cells from a population of cells. For example it may bedesirable to eliminate undifferentiated pluripotent stem cells from amixed population of cells comprising progenitor cells. In certainembodiments the method comprises contacting the population of cells witha peptide that binds specifically to the progenitor cells in thepopulation, but not to the pluripotent stem cells in the population, andseparating the peptide bound progenitor cells from the population,thereby eliminating the pluripotent stem cells from the population ofcells.

The method may be performed using any media known in the art forculturing progenitor cells. Suitable media includes commerciallyavailable cell culture media such as DMEM, MEM, RPMI, Hams Media, Media199 StemPro™ mTESR, Neuralbasal media, Smooth Muscle Cell Media, and thelike. In other embodiments the method may be practiced in a suitablebuffer such as PBS. The media may be supplemented with serum such as FBSor a serum replacement such as KOSR or B27. The media may optionallyinclude additives such as non-essential amino acids, glutamine,antibiotics such as penicillin and/or streptomycin and the like. Themethod may be performed in any suitable culture flask, e.g. a plasticflask such as a T25, T75, T150, a roller bottle or stir flask.Alternatively, the method may be performed in a multiwell plate. In someembodiments the progenitor cells may be cultured attached directly to aculture vessel such as plastic culture dish. In other embodiments theprogenitor cells may adhere to a matrix comprising one or more proteins,such as one or more proteins found in an extracellular matrix. Whengrown adherently cells may be seeded at about 30% confluency, about 40%confluency, about 50% confluency, about 60% confluency, about 70%confluency, about 80% confluency, about 90% confluency, or about 95%confluency to practice the method of the invention. The method may beperformed at about 37° C., at about 4° C. or at ambient temperature. Theprogenitor cells may be incubated with the peptide for about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours about 6hours, about 7 hours, about 8 hours or more. In some embodiments theprogenitor cell is incubated with the peptide for less than 25 hours,less than 20 hours, less than 15 hours, less than 10 hours, less than 5hours, less than 2 hours. In other embodiments the progenitor cells areincubated with the peptide for 30 minutes. In still other embodimentsthe progenitor cells are incubated with the peptide for an hour. In yetother embodiments the progenitor cells are incubated with the peptidefor 2 hours.

In some embodiments the peptide may comprise a detectable substance andthe separation may be based on detection of the detectable substance.For example the detectable substance may be a dye, such as fluorescentdye, a quantum dot (Qdot) and the peptide bound cells may be separatedusing an method known in the art including cell sorting using a flowcytometer.

In some embodiments the peptide may be a fusion protein. In someembodiments the method may further comprise contacting the peptide boundto the progenitor cell with a second molecule that specifically binds tothe peptide. For example, the second molecule that specifically binds tothe peptide may include an antibody that binds specifically to thepeptide. Where the peptide is a fusion protein comprising a moietylinked to the peptide, the second molecule may be a binding partner ofthe moiety linked to the peptide. For example, where the peptide islinked to a biotin molecule, the second molecule that binds to thefusion protein, i.e. the peptide-biotin complex, may be a streptavidinmolecule. In other embodiments, a streptavidin molecule may be bound tothe peptide and the second molecule that binds to the fusion protein,i.e. the peptide-streptavidin complex may be biotin.

In some embodiments the peptide may be a monomer. In some embodimentsthe peptide may be a multimer. In some embodiments the multivalentpeptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more peptidescapable of binding to a progenitor cell. In other embodiments themultivalent peptide comprises less than 500 peptides, less than 400peptides, less than 300 hundred peptides, less than 200 peptides, lessthan 100 peptides, less than 50 peptides, less than 25 peptides, lessthan 5 peptides capable of specifically binding to the progenitor cells.

Peptide bound progenitor cells may be separated or sorted from theremainder of the population of cells using any technique known in theart. For example, the peptide bound progenitor cells may be separated byflow cytometry, immune-preciptiation, filtration, chromatography,magnetic bead separation, density gradient and the like.

Enriched Progenitor Cell Populations

In certain embodiments the invention provides enriched populations ofprogenitor cells. The enriched population of progenitor cells may beused as therapeutic to treat a subject in need of cell replacementtherapy. For example where the enriched cell population comprisesdopaminergic neurons, the enriched population may be used to treat asubject having Parkinson's disease. Where the enriched cell populationcomprises cells producing insulin, the enriched cell population may beused to treat a subject having diabetes. Where the enriched cellpopulation includes a cell producing collagen 2, the enriched cellpopulation may be used to treat a subject having arthritis. Thus askilled artisan would appreciate that therapeutic applications willdepend on the gene expression profile of the enriched cell population.The skilled artisan would further appreciate that many other therapeuticapplications are also contemplated.

The enriched population may be used to screen for agents thatdifferentiate the progenitor cells into a more differentiated or maturephenotype. The enriched population of progenitor cells may be used indrug screening and toxicity assays. The enriched population of cells maybe used as an immunogen to generate antibodies against one or moreproteins expressed on the progenitor cell surface. Where the enrichedpopulation of cells is derived from an individual having a disease theenriched population of progenitor cells may be used to study the onsetof the disease. For example an iPS cell may be generated from anindividual having a disease such as diabetes. The iPS cell could then beused to generate an endodermal progenitor cell, capable ofdifferentiating further into an insulin producing (3-islet cell of thepancreas. The progenitor cell could be enriched according the methodsdescribed infra and used to study the onset of the diabetic phenotypeunder a variety of conditions.

In some embodiments the invention provides an enriched population ofprogenitor cells that is essentially pure. In other embodiments theinvention provides an enriched population of progenitor cells that isabout 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 95% about 99% pure. In some embodiments theinvention provides an enriched population of cells that is greater than10%, greater than 20%, greater than 30%, greater than 40%, greater than50%, greater than 60%, greater than 70%, greater than 80%, greater than90%, greater than 95%, greater than 99% pure.

In some embodiments the invention provides a population of progenitorcells that is essentially free of pluripotent stem cells. In otherembodiments the invention provides an enriched population of progenitorcells comprising about 50%, about 40%, about 30%, about 20%, about 10%,about 5%, about 1% pluripotent stem cells. In some embodiments theinvention provides an enriched population of progenitor cells comprisingless than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, less than 1% pluripotent stem cells.

Peptides

In various embodiments the invention provides peptides that specificallybind to a progenitor cell. Suitable peptides include any peptide havingspecific binding activity with respect to at least one progenitor cellor cell line. The peptide may comprise a plurality of amino acids linkedtogether by one or more peptide bonds. The peptide may have a length ofabout 3-60 amino acids, about 5-50 amino acids, about 8-35 amino acids,about 10-25 amino acids. In some embodiments the peptide is about 5,about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 45, about 50, about 55, about 60 amino acids long. In someembodiments the peptide is less than 100 amino acids long, less than 90amino acids long, less than 80 amino acids long, less than 70 aminoacids long, less than 60 amino acids long, less than 50 amino acidslong, less than 40 amino acids long, less than 30 amino acids long, lessthan 20 amino acids long, less than 10 amino acids long. In someembodiments the peptide is about 12 amino acids long. In someembodiments the peptide is 12 amino acids long.

In some embodiments the peptide may be antibody or a fragment of anantibody. In other embodiments the peptide is not an antibody or afragment of an antibody. In some embodiments the peptide comprises anamino acid sequence having PLEXIN homology. In some embodiments theamino acid is substantially homologous to PLEXIN. In some embodimentsthe peptide binds specifically to one or more ligands, such as aprotein, a carbohydrate or a lipid expressed on an endoderm cell. Inother embodiments the peptide binds specifically to one or more ligands,such as a protein, a carbohydrate or a lipid expressed on a mesodermcell. In some embodiments the peptide binds specifically to one or moreligands, such as a protein, a carbohydrate or a lipid expressed on asmooth muscle cell. In other embodiments the peptide binds specificallyto one or more ligands, such as a protein, a carbohydrate or a lipidexpressed on W10 cell.

In some embodiments the invention provides a peptide selected fromSWTYSYPNQNMD (SEQ ID NO: 1); DWTYSLPGLVEE (SEQ ID NO: 2);NWTWSMPTGNPA(SEQ ID NO: 3); GMTLRVLTN-YTE- (SEQ ID NO: 4); TLHVSENSWTYN(SEQ ID NO: 5); DWLWSFAPNVDT (SEQ ID NO: 6); TLSSQNPYMHKK (SEQ ID NO:7); IDKQMMTSHKAI (SEQ ID NO: 8); QGMETQKLRMLK (SEQ ID NO: 9);GWYWETPLDMFN (SEQ ID NO: 10); GWVIDYDYYPMR (SEQ ID NO: 11); VTAENYQSFSVS(SEQ ID NO: 12); NNKMDDRMMMSIV (SEQ ID NO: 13); STGTDLHSNARI (SEQ ID NO:14); YEFDNLLNRTLW (SEQ ID NO: 15); EWTVNERTMWDL (SEQ ID NO: 16) andseparating the progenitor cell bound to the peptide comprising the aminoacid sequence chosen from SWTYSYPNQNMD (SEQ ID NO: 1); DWTYSLPGLVEE (SEQID NO: 2); NWTWSMPTGNPA(SEQ ID NO: 3); GMTLRVLTN-YTE- (SEQ ID NO: 4);TLHVSENSWTYN (SEQ ID NO: 5); DWLWSFAPNVDT (SEQ ID NO: 6); TLSSQNPYMHKK(SEQ ID NO: 7); IDKQMMTSHKAI (SEQ ID NO: 8); QGMETQKLRMLK (SEQ ID NO:9); GWYWETPLDMFN (SEQ ID NO: 10); GWVIDYDYYPMR (SEQ ID NO: 11);VTAENYQSFSVS (SEQ ID NO: 12); NNKMDDRMMMSIV (SEQ ID NO: 13);STGTDLHSNARI (SEQ ID NO: 14); YEFDNLLNRTLW (SEQ ID NO: 15); EWTVNERTMWDL(SEQ ID NO: 16). Also contemplated are variants of the above identifiedsequences. The variants may comprise one or more conservative amino acidsubstitutions as described infra. Contemplated peptides include thesequences listed above wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11,amino acids identified in any of the sequences have been substitutedwith a non-identical conservative amino acid substitution, provided thatthe peptide can still specifically bind a target molecule on the sameprogenitor cell as original sequence.

In some embodiments the peptides of the invention have one or moreconserved consensus sequences that identify the peptide as a peptidethat specifically binds to a progenitor cell. The consensus sequence maybe chosen from amino acid sequences DWLW-(SEQ ID NO: 22), -DLWSF- (SEQID NO: 23), -WLW-, -WLWS- F-P (SEQ ID NO: 24), -PNV-, F-PNV- (SEQ ID NO:25), -SF-, -SF-PNV- (SEQ ID NO: 26), -PNV-T- (SEQ ID NO: 27), -W-W-,WLWSF-P (SEQ ID NO: 28), -Y-F-, -NLLN- (SEQ ID NO: 29), Y-F-NLLN-(SEQ IDNO: 30), -LL-RT-(SEQ ID NO: 31), -NRTL-(SEQ ID NO: 32), -NLLNRTL-(SEQ IDNO: 33), F-NLLNRTLA-(SEQ ID NO: 34), and Y-F-NLLNRTL(SEQ ID NO: 35). Inother embodiments the invention provides a peptide that selectivelybinds to a progenitor cell wherein the peptide comprises an amino acidsequence chosen from DWLW- (SEQ ID NO: 22), -DLWSF-(SEQ ID NO: 23)-WLW-, -WLWS- F-P (SEQ ID NO: 24), -PNV-, F-PNV- (SEQ ID NO: 25), -SF-,-SF-PNV- (SEQ ID NO: 26), -PNV-T- (SEQ ID NO: 27), -W-W-, WLWSF-P (SEQID NO: 28), -Y-F-, -NLLN- (SEQ ID NO: 29), Y-F-NLLN- (SEQ ID NO: 30),-LL-RT- (SEQ ID NO: 31), -NRTL-(SEQ ID NO: 32), -NLLNRTL- (SEQ ID NO:33), F-NLLNRTLA- (SEQ ID NO: 34), Y-F-NLLNRTL (SEQ ID NO: 35).

Also contemplated are peptides having amino acid sequence identified inthe preceding paragraph wherein one or more amino acids have beenmutated while still maintaining biological activity, e.g. the ability tospecifically bind to a progenitor cell. In some embodiments 1, 2, 3, 4,5 or more of the amino acids have been mutated. Mutations can beintroduced into any of the sequences identified above, for example, bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. For example, conservative amino acid substitutions may bemade at one or more predicted non-essential amino acid residues. Anon-essential amino acid is one that is not required to maintainbiological activity. In some embodiments one or more essential aminoacids may be mutated, e.g. by substituting a conservative amino acid forthe native amino acid.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential or essential amino acid residue in the peptide isreplaced with another amino acid residue from the same side chain familyAlternatively, in another embodiment, mutations can be introducedrandomly along all or part of a peptide coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forprogenitor cell binding activity to identify mutants that retainactivity. Following mutagenesis of any of the peptides can be expressedby any recombinant technology known in the art and the activity of therecombinant protein can be determined.

In some embodiments the peptide comprises a detectable substance. Thedetectable substance may be linked to the peptide. For example thedetectable substance may be covalently linked to the peptide ornon-covalently linked to the peptide. In some embodiments the detectablesubstance is linked to the carboxy terminus of the peptide. In otherembodiments the detectable substance may be linked to the amino terminusof the peptide. In still other embodiments the detectable substance maybe linked to both the amino terminus and the carboxy terminus of thepeptide. In certain embodiments the peptide may be linked to one or moreR groups or side chains of one or more amino acids comprising thepeptide. In still further embodiments the peptide may be linked to acombination of one or more R groups of one or more amino acidscomprising the peptide and the amino terminus of the peptide and/or thecarboxy terminus of the peptide.

Suitable detectable substances include any substance which can bevisually detected, e.g. a fluorescent or luminescent substance, or anysubstance that can be detected by using some detecting means, e.g. aradioactive label, a member of a specific binding pair, e.g. a nucleicacid sequence, hapten, etc.

Any fluorescent, luminescent, bioluminescent or radioactive moleculesmay be used as the labels. Many of them are commercially available, forexample fluorescent stains Alexa Fluors (Molecular Probes) and DyLightFluors (Thermo Fisher Scientific). Other non-limited examples offluorescent labels may be the following molecules: Fluoresceinisothiocyanate (FITC), 5-(and 6)-carboxyfluorescein, 5- or6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoicacid, fluorescein isothiocyanate, rhodamine, DAPI, tetramethylrhodamine,Cy2, Cy3, Cy5, AMCA, PerCP, R-phycoerythrin (RPE) allophycoerythrin(APC), Texas Red, Princeton Red, Green fluorescent protein (GFP) coatedCdSe nanocrystallites, ruthenium derivatives, luminol, isoluminol,acridinium esters, 1,2-dioxetanes and pyridopyridazines, radioactiveisotopes of hydrogen, carbon, sulfur, iodide, cobalt, selenium, tritium,or phosphor.

In some embodiments the detectable label may be an enzyme. Non-limitingexamples of suitable enzyme labels may be alkaline phosphatase (AP),beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase,beta-N-acetylglucosaminidase, beta.-glucuronidase, invertase, xanthineoxidase, firefly luciferase, glucose oxidase (GO). Such enzymes may beused in combination with a chromogen, a fluorogenic compound or aluminogenic compound to generate a detectable signal.

In other embodiments, the detectable label may be a member of a specificbinding pair, e.g. a hapten. As non-limiting examples of suitablehaptens may be mentioned 2,4-dinitrophenol (DNP), digoxigenin,fluorescein, Texas Red, tetra methyl rhodamine, nitrotyrosine,acetylaminofluorene, mercury trintrophonol, estradiol, bromodeoxyuridine, dimethylaminonaphthalene sulfonate (dansyl); examples ofsuitable specific binding pairs may include biotin, streptavidin,complementary natural and non-natural oligonucleotide sequences, zincfingers binding domain pairs, etc. In certain embodiments greenfluorescent protein (GFP) may be used, for example where a peptide isrecombinantly produced the GFP protein can be engineered into thepeptide.

In some embodiments, the detectable substance may be a biotag. Biotagsdescribed herein include a reporter binding domain to provide a bindingsite for the reporter. For example, when the reporter or diagnosticagent is a metal (e.g., a noble metal or superparamagnetic metal) orparamagnetic ion, the biotag may include a metal binding domain. In suchcase, the reporter may be reacted with a reagent having a long tail withone or more chelating groups attached to the long tail for binding theseions. The long tail may be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chain havingpendant groups to which may be bound to a chelating group for bindingthe ions. Examples of chelating groups that may be used according to thedisclosure include, but are not limited to, ethylenediaminetetraaceticacid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA,NETA, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones,polyoximes, and like groups. The chelate is normally linked to the PSMAantibody or functional antibody fragment by a group which enablesformation of a bond to the molecule with minimal loss ofimmunoreactivity and minimal aggregation and/or internal cross-linking.The same chelates, when complexed with non-radioactive metals, such asmanganese, iron and gadolinium are useful for MRI, when used along withthe antibodies and carriers described herein. Macrocyclic chelates suchas NOTA, DOTA, and TETA are of use with a variety of metals andradiometals including, but not limited to, radionuclides of gallium,yttrium and copper, respectively. Other ring-type chelates such asmacrocyclic polyethers, which are of interest for stably bindingnuclides, such as ²²³Ra for RAIT may be used. In certain embodiments,chelating moieties may be used to attach a PET imaging agent, such as anAl¹⁸F complex, to a targeting molecule for use in PET analysis.

The peptide may be conjugated to a Quantum Dot. Quantum dots are tinyparticles, or “nanoparticles”, of a semiconductor material,traditionally chalcogenides (selenides or sulfides) of metals likecadmium or zinc (CdSe or ZnS, for example), which range from 2 to 10nanometers in diameter. Because of their small size, quantum dotsdisplay unique optical and electrical properties that are different incharacter to those of the corresponding bulk material. The mostimmediately apparent of these is the emission of photons underexcitation, which are visible to the human eye as light. Moreover, thewavelength of these photon emissions depends not on the material fromwhich the quantum dot is made, but its size. The ability to preciselycontrol the size of a quantum dot enables the manufacturer to determinethe wavelength of the emission, which in turn determines the color oflight the human eye perceives. Quantum dots can therefore be “tuned”during production to emit any color of light desired. The ability tocontrol, or “tune” the emission from the quantum dot by changing itscore size is called the “size quantization effect”. The smaller the dot,the closer it is to the blue end of the spectrum, and the larger thedot, the closer to the red end. Dots can also be tuned beyond visiblelight, into the infra-red or into the ultra-violet.

The number or detectable labels per reporter molecule may vary. In someembodiments 1 to 3, for example 1, 2 or 3 labels per peptide may beused. In one embodiment the peptide comprises one detectable label. Inanother embodiment the peptide comprises two to four residues which aredetectable substances. In some embodiments, the peptide may comprisemore than 3 labels, such as 4 to 150 labels per peptide molecule, 10 to100 labels per peptide molecule, 20-80 labels per peptide molecule,30-60 labels per peptide molecule, 40-50 labels per peptide molecule,1-10 labels per peptide molecule, 2-8 labels per peptide molecule, 3-6labels per peptide molecule.

Peptide Fusion Proteins

The invention also provides peptide chimeric or fusion proteins. As usedherein, a peptide “chimeric protein” or “fusion protein” comprises apeptide as described infra operatively linked to a second peptide orprotein. Within a peptide fusion protein the peptide can correspond toall or a portion of one or more of the peptide sequences disclosedinfra. In one embodiment, a peptide fusion protein comprises at leastone biologically active portion of a peptide disclosed infra. Abiologically active portion of a peptide may refer to the portion of thepeptide that specifically binds to a progenitor cell. In anotherembodiment, a peptide fusion protein comprises at least two biologicallyactive portions of a peptide disclosed infra. In yet another embodimenta peptide fusion protein comprises at least three biologically activeportions of a peptide disclosed infra. Within the fusion protein, theterm “operatively linked” is intended to indicate that the peptide thatbinds to a progenitor cell and the second peptide or protein are linkedin-frame to each other. The second peptide or protein can be fused tothe N-terminus or C-terminus of the peptide that binds to the progenitorcell. The second peptide or protein may be, for example, the Fc portionof an antibody. This may be operatively joined to either the N-terminusor the C-terminus of the peptide that binds to the progenitor cell.Fc-target protein fusions have been described in Lo et al. (1998)Protein Engineering 11:495-500, and U.S. Pat. Nos. 5,541,087 and5,726,044.

In one embodiment a peptide fusion protein comprises a progenitor cellbinding domain operably linked to the extracellular domain of a secondprotein or peptide. Such fusion proteins can be further utilized inscreening assays for compounds which modulate the peptide bindingactivity. It is also contemplated that the peptide fusion protein may beused to facilitate isolation or enrichment of the progenitor cell whichbinds to the peptide. Also contemplated is the use of the fusion peptideto isolate or enrich for the peptide having the biological activity ofbinding to the progenitor cell.

In another embodiment, the fusion protein is a GST-peptide fusionprotein in which the peptide binds specifically to a progenitor cellwherein the peptide sequences are fused to the C-terminus of the GST(i.e., glutathione S-transferase) sequences. Such fusion proteins canfacilitate the purification of a recombinant peptide and/or theenrichment of progenitor cells bound to the fusion protein.

In another embodiment, the fusion protein is a protein containing aheterologous signal sequence at its N-terminus. For example, the peptidemay comprise a signal sequence, fused to the 5′ end of the peptidecoding sequence for efficient secretion of the peptide fusion proteinwhere the peptide is produced using recombinant technology. Expressionand/or secretion of the peptide can be increased through use ofdifferent heterologous signal sequences.

In yet another embodiment, the fusion protein is apeptide-immunoglobulin fusion protein in which the peptide sequencescomprising one or more domains are fused to sequences derived from amember of the immunoglobulin protein family The peptide-immunoglobulinfusion proteins can be used to affect the bioavailability of a peptidecognate ligand. Moreover, the peptide-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-peptideantibodies in a subject, to purify peptide ligands, and in screeningassays to identify molecules that inhibit the interaction of the peptidewith a peptide ligand. The peptide immunoglobulin fusion protein can beused to separate and/or enrich for progenitor cells bound to the peptideimmunoglobulin fusion protein.

A peptide chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. Eds. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A peptide-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to thepeptide.

Methods of Making Peptides

The peptides of the instant invention may be made according to anymethod known in the art. In some embodiments the peptides are producedrecombinantly, e.g. by transfecting a cell with a nucleic acid encodingthe peptide into a host cell and expressing the peptide in the hostcell. The nucleic acid may be for example, DNA, or RNA. In someembodiments the nucleic acid encoding the peptide is transfected intothe host cell in a vector. The vector may be for example, a plasmid,many of which are commercially available or a viral vector, which arealso commercially available. The cell may be a prokaryotic cell such asE. coli or a eukaryotic cell such as a mammalian cell, e.g., HeLa cell,or a COS cell, insect cells such as SF9 cells, yeast cells such as Piciapastoris, or the like. In some embodiments the invention thus provides acell transfected with a nucleic acid encoding a peptide of theinvention.

In other embodiments the peptide may be chemically synthesized. Anyknown method of synthesizing a peptide may be used. A number oftraditional techniques for chemically synthesizing proteins, such assolid phase synthesis are known in the art, see, e.g., Merrifield, 1973,Chemical Polypeptides, (Katsoyannis and Panayotis eds.) pp. 335-61;Merrifield 1963, J. Am. Chem. Soc. 85:2149; Davis et al. 1985, Biochem.Intl. 10:394; Finn et al. 1976, The Proteins (3d ed.) 2:105; Erikson etal. 1976, The Proteins (3d ed.) 2:257; U.S. Pat. No. 3,941,763.

Improvements in the chemical synthesis of proteins include the advent ofnative chemical ligation. As initially described, native ligationprovides for the rapid synthesis of large polypeptides with a naturalpeptide backbone via the native chemical ligation of two or moreunprotected peptide segments. In native ligation none of the reactivefunctionalities on the peptide segments need to be temporarily masked bya protecting group. Native ligation also allows for the solid phasesequential chemical ligation of peptide segments in an N-terminus toC-terminus direction, with the first solid phase-bound unprotectedpeptide segment bearing a C-terminal alpha-thioester that reacts withanother unprotected peptide segment containing an N-terminal cysteine.Native chemical ligation also permits the solid-phase ligation in the C-to N-terminus direction, with temporary protection of N-terminalcysteine residues on an incoming (second) peptide segment (see, e.g.,U.S. Pat. No.: 6,326,468; WO 02/18417). Native ligation may also becombined with recombinant technology using intein linked to a chitinbinding domain (Muir et al., 1998, Proc. Natl. Acad. Sci. USA, 95:6705).

Thus in certain embodiments the invention provides a synthetic peptide,i.e. a peptide produced in vitro by chemical synthesis that specificallybinds to a progenitor cell.

Progenitor Cells

In certain embodiments the progenitor cells are multipotent cells. Incertain embodiments the progenitor cells are not pluripotent cells.

Progenitor cells derived from pluripotent cells, such as human embryonicstem cells have been described (West et al. (2008) Regen Med. 3(3):287).The progenitor cells will be essentially genetically identical to theparental stem cell population from which they originated. Thus theprogenitor cell may be about 95%, about 96%, about 97%, about 98%, about99% genetically identical to the pluripotent stem cell from which it wasdifferentiated. In some embodiments the progenitor cell may be greaterthan 95%, greater than 96%, greater than 97%, greater than 98%, greaterthan 99% genetically identical to the pluripotent stem cell it wasdifferentiated from. Because the progenitor cell is essentiallygenetically identical to its parental pluripotent stem cell and becausethe parental pluripotent stem cell can be cultured almost indefinitely,the invention provides a limitless supply of a progenitor cell having aspecific genomic makeup.

Progenitor cells may be obtained by differentiating a pluripotent stemcell in vitro using defined set of culture conditions. Progenitor cellscan be obtained using the method described by West, Id. Briefly ashotgun approach is used that comprises a two-step protocol. In thefirst step pluripotent stem cells, such as hES cells, are differentiatedunder an array of in vitro conditions that include colony in situdifferentiation, differentiation as embryoid bodies, on non-adherentplastic or hanging drops and differentiation of in the presence ofgrowth factors for various periods of time. The resultant cultures inthis matrix are designated candidate cultures (CC). Althoughheterogeneous, these CCs are enriched for particular cell types. Each CCmay be plated at a clonal density in an array of different cell culturemedia optimized for various stromal and epithelial cell types. Thecultures can be allowed to grow in 5% ambient oxygen for a suitableperiod of time, e.g. 14 days.

Progenitor cells are partially differentiated endodermal, ectodermal andmesodermal cell types that have not undergone terminal differentiation.Typically they are clonal, meaning that they are grown as a colonyderived either from a single parental cell. Alternatively, the colony ofprogenitor cells may be derived from a small number of parental cells.They may express some genes generally seen during embryonic developmentsuggesting that they are an early progenitor of mature cell types foundin the adult. Thus in some embodiments of the invention the progenitorcells express one or more of the embryonic genes chosen from MEOX1,MEOX2, FOXF1, ENC1, LXH8, ROR2, SHOX2, GPC2, HSPG and FOXC1. In someembodiments of the invention the progenitor cells express CD133 and/orCD24. In some embodiments of the invention the progenitor cells do notexpress markers found on pluripotent stem cells such as OCT4. In someembodiments the progenitor cells express lower levels of EBAF, ZNF206and ZIC3 compared to embryonic stem cells.

In some embodiments the progenitor cells express genes found inneuroglial cells such as one or more of the genes chosen from PLP1,PMP2, GRIN1 and GABRA1. In some embodiments the progenitor cells expressa gene expressed in the transport of secretory vesicles found in neuronsand melanocytes, e.g. Myosin Va. In other embodiments the progenitorcells express a gene expressed in limb dermis, smooth muscle andvascular endothelial cells, e.g. GARP. In certain embodiments theprogenitor cells express a gene associated with the regulation ofvascular morphogenesis, e.g., EDIL3. In some embodiments the progenitorcells express a gene expressed in bone and cornea, e.g. COL24A1. In someembodiments the progenitor cells express a gene expressed byoligodendrocytes, e.g. SEMA5A.

In certain embodiments the progenitor cells express one or more homeoboxgenes chosen from DLX, MEOX, HOX, LIM, MSX, BAPX, PRRX, GSC, IRX, SOX,PITX and FOX. In some embodiments the progenitor cells do not formteratomas when injected into scid mice, but nonetheless express one ormore oncofetal genes chosen from PLAG1, AMIGO2, HCLS1, SPINK1, PRAME,INSM1, RAGE, ENC1, BCAS1, GRM1, TSGA10, S100A2, A4, A6, GPC3, EGFL6,PSG5, CEACAM1, CGPC3, SRPUL, DCDC2, LRRN5, SOX11, RUNX3, CA12, STARD10,CXCL1, ANPEP, GAGE6, NCOA6, TACSTD2, and TSPAN8.

Progenitor cells maintain the potential to differentiate further intoterminally differentiated cell types. Progenitor cells may have telomerelengths that greater than what is ordinarily found in a terminallydifferentiated cell. Typically, because of their replicative capacity,they are capable of being produced on a large scale. Generally, they donot form teratomas when transplanted into immunocompromised mice,confirming that they are no longer pluripotent.

Any progenitor cell known in the art may be used in the methods andcompositions described infra (See, e.g. US Patent ApplicationPublication Nos: 20080070303; 20100184033; 20120171171). In someembodiments the progenitor cell is a cell that is no longer pluripotent,but still retains the potential to differentiate further into at leastone type of mature cell. The progenitor cell may be clonal, such that itis substantially free of any other contaminating cell type.Alternatively, the progenitor cell may be one of several cell typesfound in vitro cell population.

In some embodiments the progenitor cell used for the various embodimentsdescribed infra may be chosen from any of the following establishedprogenitor cell lines, including, but not limited to W10, 4D20.8, SM30,7PEND24, E15. In certain embodiments the progenitor cell line is W10. Inother embodiment the progenitor cell lines may be chosen from one of thefollowing progenitor cell lines: B16, B28, 6-1, B26, B11, B2, CM02, E75,E15, E30, E3, E73, E57, E67 4D20.9, E72, EN7, En55, SK17, Z11, E68,E109, ELS5.8, and M10.

The progenitor cell may have the ability to proliferate in culture overan extensive period of time. For example, a progenitor cell may bepassaged in culture about 100 times, about 90 times, about 80 times,about 70 times, about 60 times, about 50 times, about 40 times, about 30times about 20 times, about 10 times, about 5 times. In some embodimentsthe progenitor cell may be passaged in culture about 1-60 times, about5-50 times, about 10-40 times, about 20-30 times.

Methods of Detecting Progenitor Cells

In some embodiments the invention provides a method of detecting aprogenitor that is bound to a peptide that specifically binds to theprogenitor cell. The peptide may comprise a detectable substance, asdescribed infra, to facilitate detection of the cell peptide boundcomplex. The cell may be detected in vitro, e.g. by flow cytometry,immunocytochemistry (e.g., staining with tissue specific or cell-markerspecific antibodies), fluorescence activated cell sorting (FACS),magnetic activated cell sorting (MACS), fluorescent microscopy, ELISA,radio-immuno-assay, western blot, autoradiography and the like. Peptidebound progenitor cells may be detected in vivo, e.g. by photon laserscanning microscopy, X-Ray, CT Raman, MRI, USG and NMR.

Methods of Monitoring the Differentiation of Progenitor Cells

In certain embodiments the invention provides a method of monitoring thedifferentiation of a progenitor cell. The progenitor cell may bemonitored for differentiation in vitro or in vivo. The progenitor cellmay be contacted with one or more peptides that specifically bind to theprogenitor cell. The one or more peptides may comprise a detectablesubstance. The one or more peptides may be provided as monovalent or amulti-valent composition. The peptide comprising the detectablesubstance bound to the progenitor cell may be monitored/detected over aperiod of time to allow for the detection of the differentiation of theprogenitor cell over time. Any suitable assay may be used to monitor theprogenitor cell over time and thereby detect the differentiation of theprogenitor cell. Cells may be monitored over time using, for example,microscopy, such as fluorescent microscopy, flow cytometry,immunofluorescence, cytohistochemistry, RIA and ELISA. In someembodiments gene expression may be analyzed to monitor thedifferentiation of the progenitor cell. For example PCR, Southern orwestern blots may be used.

Methods of Screening for Peptides

In certain embodiments the invention provides a method of screening forpeptides that bind to a progenitor cell. The method may comprise 1)contacting a progenitor cell with a candidate peptide, 2) washing theprogenitor cell from step 1) and detecting the bound peptide from step2). Any detection method described known in the art, including thosedescribed infra may be used.

In some embodiments, the screening method may rely on phage display.Phage display is used for the high-throughput screening of proteininteractions. In the case of M13 filamentous phage display, the DNAencoding the protein or peptide of interest is ligated into the pIII orpVIII gene, encoding either the minor or major coat protein,respectively. Multiple cloning sites may be used to ensure that thefragments are inserted in all three possible reading frames so that thecDNA fragment is translated in the proper frame. The phage gene andinsert DNA hybrid is then transformed into Escherichia coli (E. coli)bacterial cells such as TG1, SS320, ER2738, or XL1-Blue E. coli. If a“phagemid” vector is used (a simplified display construct vector) phageparticles will not be released from the E. coli cells until they areinfected with helper phage, which enables packaging of the phage DNA andassembly of the mature virions with the relevant protein fragment aspart of their outer coat on either the minor (pIII) or major (pVIII)coat protein. By contacting a relevant target, such as a progenitorcell, with the transformed phage, a phage that displays a protein thatbinds to one of those targets on its surface will bind while others maybe removed by washing. Those that remain can be eluted, used to producemore phage (by bacterial infection with helper phage) and so produce aphage mixture that is enriched with relevant (i.e. binding) phage. Therepeated cycling of these steps is referred to as ‘panning’ Phage elutedin the final step can be used to infect a suitable bacterial host, fromwhich the phagemids can be collected and the relevant DNA sequenceexcised and sequenced to identify the relevant, interacting proteins orprotein fragments. A library of DNA sequences can be screened in thismanner to identify peptide sequences that bind to the progenitor cell.Phage libraries may be generated using techniques known in the art.Alternatively, phage libraries may be purchased from a commercial vendor(e.g. New England BioLabs, Ipswich, Mass.).

Peptide Progenitor Cell Complexes

The invention provides compositions comprising a peptide and aprogenitor cell. The progenitor cell may be any progenitor celldescribed infra, e.g. an endoderm progenitor cell, a mesoderm progenitorcell. In some embodiments the composition comprising the peptide andprogenitor cell is isolated from other cell types. In some embodimentsthe composition comprising a peptide progenitor cell is enriched for theprogenitor cell and peptide. In some embodiments the compositioncomprising the peptide and the progenitor cell is essentially free ofpluripotent stem cells. In some embodiments the composition comprisingthe peptide and the progenitor cell has been depleted of pluripotentstem cells. In some embodiments the composition comprising peptide andthe progenitor cell may be present in a population of cells comprising aplurality of cell types. In some embodiments the composition comprisingthe peptide and the progenitor cell is an in vitro composition. In otherembodiments the composition comprising the peptide and the progenitorcell is an in vivo composition.

The peptide may be bound, e.g. chemically bound, to the progenitor cell.The peptide may be covalently bound to the progenitor cell. The peptidemay be covalently bound to a protein, carbohydrate or lipid expressed onthe surface of the progenitor cell. In some embodiments the peptide maybe taken up or internalized, e.g. by endocytosis, by the progenitorcell. The peptide may be any peptide described infra. The peptide mayfurther comprise one or more detectable substances. In some embodimentsthe peptide may be non-covalently bound to the progenitor cell. Forexample the peptide may be bound to the cell by an ionic interaction, ahydrophobic interaction, a hydrophilic interaction or the like.

In certain embodiments the invention provides a composition comprising aprogenitor cell and one or more peptides bound the progenitor cell. Oneor more peptides may include about 1, about 2, about 4, about 8, about16, about 32, about 64, about 128, about 200, about 400, about 500peptides. In some embodiments a plurality of peptides includes less than50,000, less than 40,000, less than 30,000, less than 20,000, less than10,000, less than 5,000, less than 1,000, less than 500, less than 100,less than 50 peptides. In some embodiments 1 peptide, 2 peptides, 5peptides, 10 peptides, 20 peptides, 100 peptides, 1000 peptides, 10,000peptides are bound to the progenitor cell in the composition comprisingthe peptide and the progenitor cell.

Pluripotent Stem Cells

Pluripotent stem cells are cells that have the potential, under theappropriate culture conditions to differentiate into cells from allthree germ layers and are capable of immortal proliferation in vitro.Their ability to differentiate into cells from all three germ layersprovides the potential to manufacture, in vitro, virtually any cell inthe body. Their ability to proliferate virtually endlessly in cultureprovides a means to scale the manufacture of cellular therapeutics byproviding an endless supply of identical starting material from whichcellular therapeutics can be manufactured. Thus, pluripotent stem cellsprovide the potential to derive cellular therapeutics to treat aplethora of otherwise intractable human diseases such as Parkinson'sdisease, diabetes, spinal cord injury, multiple sclerosis, stroke, heartdisease, osteoarthritis, macular degeneration to name but a few.

In certain embodiments pluripotent stem cells may be derived from anymammal, including primates such as humans, and macaques, rodents such asmice and rats, and ungulates such as cows and sheep, as well as pigs,horses and the like. Stem cells may be isolated from the inner cell massof the blastocyst stage of a fertilized egg, such as an in vitrofertilized egg. Typically, the pluripotent stem cells are not derivedfrom a malignant source. Pluripotent stem cells will form teratomas whenimplanted in an immuno-deficient mouse, e.g., a SCID mouse. In someembodiments the pluripotent stem cell is a human pluripotent stem cell.

Human pluripotent stem cells are characterized by the expression ofOCT-4, telomerase and alkaline phosphatase, as well as the expression ofcell surface markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and the absenceof the marker SSEA-1. Morphologically the cells have prominent nucleoli,and a high nucleus to cytoplasm ratio.

Human pluripotent stem cells may be maintained indefinitely in cultureunder suitable conditions. Exemplary conditions include a matrix forgrowing the cells on such as feeder cells e.g. murine embryonicfibroblasts, matrigel, or a synthetic surface (See, e.g. U.S. Pat. Nos.5,843,780; 6,800,480; 7,410,798; US Patent Application Publication Nos:20120220720; 20100317101).

Numerous established human pluripotent cell lines are known. EstablishedhES cell lines include, but not limited to, H1, H7, H9, H13 or H14(Thompson, (1998) Science 282:1145); hESBGN-01, hESBGN-02, hESBGN-03(BresaGen, Inc., Athens, Ga.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6(from ES Cell International, Inc., Singapore); HSF-1, HSF-6 (fromUniversity of California at San Francisco); I 3, I 3.2, I 3.3, I 4, I 6,I 6.2, J 3, J 3.2 (derived at the Technion-Israel Institute ofTechnology, Haifa, Israel); UCSF-1 and UCSF-2 (Genbacev et al., (2005)Fertil. Steril. 83(5):1517); lines HUES 1-17 (Cowan et al., (2004) NEJM350(13):1353); and line ACT-14 (Klimanskaya et al., (2005) Lancet,365(9471):1636). Many of these cell lines are available from establishedcell banks, such as WiCell and the UK Cell Bank. cGMP qualified humanpluripotent stem cell lines including ESI 017, ESI 035, ESI 049, ESI051, and ESI 053, are available commercially from BioTime, Inc. Alameda,Calif.

pPS cells used in the present invention may have been derived in afeeder-free manner (see, e.g., Klimanskaya et al., (2005) Lancet365(9471):1636). In certain embodiments the pPS may be cultured prior touse in a serum free environment.

pPS cells may be cultured using a variety of substrates, media, andother supplements and factors known in the art. In some embodiments asuitable substrate may be comprised of a matrix including one or more ofthe following: laminin, collagen, fibronectin, vitronectin, heparinsulfate proteoglycan. In some embodiments the matrix may comprise asoluble extract of the basement membrane from a murine EHS sarcoma whichis commercially available as Matrigel™. (BD Biosciences, San Jose,Calif.). In other embodiments the matrix may comprise one more isolatedmatrix proteins of human, humanized, or murine origin, e.g., CELLstart™.(Invitrogen, Carlsbad, Calif.). In still other embodiments a suitablesubstrate may be comprised of one or more polymers such as one or moreacrylates. The polymers may include one or more proteins or peptidefragments derived from a protein found in vivo in the extra-cellularmatrix. In one particular embodiment the substrate is comprised of oneor more acrylates and a conjugated vitronectin peptide (see, e.g. U.SPatent Publication No. 2009/0191633; U.S Patent Publication No.2009/0191626; U.S Patent Publication No. 2009/0203065). pPS cells can bepropagated continuously in culture, using culture conditions thatpromote proliferation while inhibiting differentiation.

Exemplary medium may be made with 80% DMEM (such as Knock-Out DMEM,Gibco), 20% of either defined fetal bovine serum (FBS, Hyclone) or serumreplacement (US 2002/0076747 A1, Life Technologies Inc.), 1%non-essential amino acids, 1 mM L-glutamine, and 0.1 mM.beta.-mercaptoethanol. Other suitable media include serum free definedmedia such as X-VIVO™ 10 (Lonza, Walkersville, Md.). Still othercommercially available media formulations that may be used in certainembodiments of the invention include X-VIVO.™. 15 (Lonza, Walkersville,Md.); mTeSR™ (Stem Cell Technologies, Vancouver, Calif.); hTeSR∩ (StemCell Technologies, Vancouver, Calif.), StemPro™ (Invitrogen, Carlsbad,Calif.) and Cellgro™ DC (Mediatech, Inc., Manassas, Va.).

In certain embodiments, pPS cells may be maintained in anundifferentiated state without added feeder cells (see, e.g., (2004)Rosler et al., Dev. Dynam. 229:259). Feeder-free cultures are typicallysupported by a nutrient medium containing factors that promoteproliferation of the cells without differentiation (see, e.g., U.S. Pat.No. 6,800,480). In certain embodiments, conditioned media containingsuch factors may be used. Conditioned media may be obtained by culturingthe media with cells secreting such factors. Suitable cells includeirradiated (.about.4,000 rad) primary mouse embryonic fibroblasts,telomerized mouse fibroblasts, or fibroblast-like cells derived from pPScells (U.S. Pat. No. 6,642,048). Medium can be conditioned by platingthe feeders in a serum free medium such as KO DMEM supplemented with 20%serum replacement and 4 ng/mL bFGF. Medium that has been conditioned for1-2 days may be supplemented with further bFGF, and used to support pPScell culture for 1-2 days (see. e.g., WO 01/51616; Xu et al., (2001)Nat. Biotechnol. 19:971).

Alternatively, fresh or non-conditioned medium can be used, which hasbeen supplemented with added factors (like a fibroblast growth factor orforskolin) that promote proliferation of the cells in anundifferentiated form. Exemplary is a base medium like X-VIVO™ 10(Lonza, Walkersville, Md.) or QBSF™-60 (Quality Biological Inc.Gaithersburg, Md.), supplemented with bFGF at 40-80 ng/mL, andoptionally containing SCF (15 ng/mL), or Flt3 ligand (75 ng/mL) (see,e.g., Xu et al., (2005) Stem Cells 23(3):315. These media formulationshave the advantage of supporting cell growth at 2-3 times the rate inother systems (see, e.g., WO 03/020920). In some embodiments pPS cellssuch as hES cells may be cultured in a media comprising bFGF and TGFβ.Suitable concentrations of bFGF include about 80 ng/ml. Suitableconcentrations of TGFβ include about 0.5 ng/ml.

In some embodiments, the pluripotent stem cells, e.g. hES cells, may beplated at >15,000 cells cm² (optimally 90,000 cm² to 170,000 cm²).Typically, enzymatic digestion may be halted before cells becomecompletely dispersed (e.g., about 5 minutes with collagenase IV). Clumpsof about 10 to about 2,000 cells may then be plated directly onto asuitable substrate without further dispersal. Alternatively, the cellsmay be harvested without enzymes before the plate reaches confluence byincubating the cells for about 5 minutes in a solution of 0.5 mM EDTA inPBS or by simply detaching the desired cells from the platemechanically, such as by scraping or isolation with a fine pipette or acell scraper. After washing from the culture vessel, the cells may beplated into a new culture without further dispersal. In a furtherillustration, confluent human embryonic stem cells cultured in theabsence of feeders may be removed from the plates by incubating with asolution of 0.05% (wt/vol) trypsin (Gibco® Carlsbad, Calif.) and 0.05 mMEDTA for 5-15 minutes at 37.degree. C. The remaining cells in the platemay be removed and the cells may be triturated into a suspensioncomprising single cells and small clusters, and then plated at densitiesof 50,000-200,000 cells cm² to promote survival and limitdifferentiation.

In certain embodiments, primate pluripotent stem cells may be culturedon a layer of feeder cells, typically fibroblasts derived from embryonicor fetal tissue (Thomson et al. (1998) Science 282:1145). In certainembodiments, those feeder cells may be derived from human or murinesource. Human feeder cells can be isolated from various human tissues orderived by differentiation of human embryonic stem cells into fibroblastcells (see, e.g., WO 01/51616) In certain embodiments, human feedercells that may be used include, but are not limited to, placentalfibroblasts (see, e.g., Genbacev et al. (2005) Fertil. Steril.83(5):1517), fallopian tube epithelial cells (see, e.g., Richards et al.(2002) Nat. Biotechnol., 20:933), foreskin fibroblasts (see, e.g., Amitet al. (2003) Biol. Reprod. 68:2150), uterine endometrial cells (see,e.g., Lee et al. (2005) Biol. Reprod. 72(1):42).

In the practice of the present invention, there are various solidsurfaces that may be used in the culturing of cells. Those solidsurfaces include, but are not limited to, standard commerciallyavailable cell culture plates such as 6-well, 24-well, 96-well, or144-well plates. Other solid surfaces include, but are not limited to,microcarriers and disks. In certain embodiments, the microcarriers maybe used in stirred-tank bioreactors for attachment of the cells. Incertain embodiments, the microcarriers are beads. Those beads come invarious forms such as Cytodex Dextran microcarrier beads with positivecharge groups to augment cell attachment, gelatin/collagen-coated beadsfor cell attachment, and macroporous microcarrier beads with differentporosities for attachment of cells. The Cytodex dextran, gelatin-coatedand the macroporous microcarrier beads are commercially available(Sigma-Aldrich, St. Louis, Mo. or Solohill Engineering Inc., Ann Arbor,Mich.). In certain embodiments, the beads are 90-200 μm in size with anarea of 350-500 cm^(2.) Beads may be composed of a variety of materialssuch as, but not limited to, glass or plastic. Disks are sold bycompanies such as New Brunswick Scientific Co, Inc. (Edison, N.J.). Incertain embodiments, the disks are Fibra-cel Disks, which arepolyester/polypropylene disks. A gram of these disks provide a surfacearea of 1200 cm².

The solid surface suitable for growing pPS cells may be made of avariety of substances including, but not limited to, glass or plasticsuch as polystyrene, polyvinylchloride, polycarbonate,polytetrafluorethylene, melinex, or thermanox. In certain embodiments ofthe invention, the solid surfaces may be three-dimensional in shape.Exemplary three-dimensional solid surfaces are described, e.g., in US2005/0031598.

In certain embodiments, the cells may be in a single-cell suspension.The single-cell suspension may comprise culturing the cells in a spinnerflask, in a shaker flask, or in a fermenters. Fermenters that may beused include, but are not limited to, Celligen Plus (New BrunswickScientific Co, Inc., Edison, N.J.), and the STR or the Stirred-TankReactor (Applikon Inc., Foster City, Calif.). In certain embodiments,the bioreactors may be continuously perfused with media or used in afed-batch mode. Other suitable bioreactors include the Wave Bioreactorbags (GE Healthcare, Piscataway, N.J.). Bioreactors come in differentsizes including, but not limited to 2.2 liter, 5 liter, 7.5 liter, 14liter or 20 liter, 100 liter, 100 liter, 10,000 liter or larger.

Induced Pluripotent Stem Cells

Progenitor cells may be obtained from an induced pluripotent stem cell(iPS). An induced pluripotent stem cell is a pluripotent cell, i.e. ithas the ability to differentiate into at least one cell type derivedfrom each of the three primary germ layers, ectoderm, endoderm andmesoderm, but the iPS cell is typically derived from a non-pluripotentcell, such as a somatic cell. Typically an iPS cell is not derived froma fertilized egg, e.g. the blastocyst of the fertilized egg. An iPS cellmay be a somatic cell, that has been genetically reprogrammed to revertback to a pluripotent state typically found in embryonic stem cells(see, e.g. US Patent Application Publication Nos: 20080233610;20090047263; 20100003757). The reprogramming can be achieved bycontacting, e.g., transfecting, the target cell with one or morereprogramming factors. The reprogramming factors may be a nucleic acid,such as DNA or RNA encoding a factor, a protein factor capable ofreprogramming the target cell, or a small molecule capable of inducingexpression of one or more reprogramming factors within a target cell.

iPS cells may be derived by transfection of certain stem cell-associatedgenes into non-pluripotent cells, such as adult fibroblasts.Transfection is typically achieved through viral vectors, such asretroviruses. Transfected genes include the master transcriptionalregulators Oct-3/4 (Pou5f1) and Sox2, although it is suggested thatother genes enhance the efficiency of induction. After 3-4 weeks, smallnumbers of transfected cells begin to become morphologically andbiochemically similar to pluripotent stem cells, and may be isolatedthrough morphological selection, doubling time, or through a reportergene and antibiotic selection.

Yamanaka et al. have successfully transformed human fibroblasts intopluripotent stem cells using the same four pivotal genes: Oct3/4, Sox2,Klf4, and c-Myc with a retroviral system (US Patent Publication No.20090047263). Thomson and colleagues used OCT4, SOX2, NANOG, and adifferent gene LIN28 using a lentiviral system (US Patent PublicationNo. 20080233610).

Potency-determining factors that can reprogram somatic cells include,but are not limited to, factors such as Oct-4, Sox2, FoxD3, UTF1,Stella, Rex1, ZNF206, Sox15, Myb12, Lin28, Nanog, DPPA2, ESG1, Otx2c-Myc and Klf4, or combinations thereof.

One of the strategies for avoiding shortcomings of reprogramming somaticcells has been to use small compounds that can mimic the effects oftranscription factors. These molecule compounds can compensate for areprogramming factor that does not effectively target the genome orfails at reprogramming for another reason; thus they raise reprogrammingefficiency. They also avoid the problem of genomic integration, which insome cases contributes to tumor genesis. Studies using this strategywere conducted in 2008. Melton et al. studied the effects of histonedeacetylase (HDAC) inhibitor valproic acid. They found that it increasedreprogramming efficiency 100-fold (compared to Yamanaka's traditionaltranscription factor method) (Huangfu et al. (2008) Nature Biotech.26:795). The researchers proposed that this compound was mimicking thesignaling that is usually caused by the transcription factor c-Myc. Asimilar type of compensation mechanism was proposed to mimic the effectsof Sox2. In 2008, Ding et al. used the inhibition of histone methyltransferase (HMT) with BIX-01294 in combination with the activation ofcalcium channels in the plasma membrane in order to increasereprogramming efficiency (Desponts et al. (2008) Cell Stem Cell 3:568).

In 2009, Ding and colleagues demonstrated a successful alternative totranscription factor reprogramming through the use of drug-likechemicals. This was the first method in human cells that wasmechanism-specific for the reprogramming process. Ding tackled theproblem of genomic insertion by using purified proteins to transformadult cells into embryonic-like cells. Efficiency was improved usingALK5 inhibitor SB431412 and MEK inhibitor PD0325901, which when used incombination were highly effective at promoting the transformation fromfibroblast to iPS cell (see, e.g. Zhou et al. (2009) Cell Stem Cell8:381; Abujarour et al. (2009) Genome Biol. 10:220; Lin et al. (209)Nature Methods 6:805).

This two-chemical technique increased the efficiency of the classicalgenetic method by 100 fold. Using Thiazovivin with the two previouschemicals, efficiency was increased by 200 fold. Furthermore, thismethod took only two weeks to complete reprogramming while the classicmethod took four weeks (see, e.g. Science Daily Dec. 10, 2009).

Induced pluripotent stem cells may express any number of pluripotentcell markers, including: alkaline phosphatase (AP); ABCG2; stagespecific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60;TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; .beta.III-tubulin;.alpha.-smooth muscle actin (.alpha.-SMA); fibroblast growth factor 4(Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296);N-acetyltransferase-1 (Nat1); (ES cell associated transcript 1 (ECAT1);ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1;ECAT15-2; Fthl17; Sal14; undifferentiated embryonic cell transcriptionfactor (Utf1); Rex1; p53; G3PDH; telomerase, including TERT; silent Xchromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15(Fbx15); Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3;Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency-associated 2(DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3/Stella; DPPA4; othergeneral markers for pluripotency, etc. Other markers can include Dnmt3L;Sox15; Stat3; Grb2; SV40 Large T Antigen; HPV16 E6; HPV16 E7,(β-catenin, and Bmi1. Such cells can also be characterized by thedown-regulation of markers characteristic of the differentiated cellfrom which the iPS cell is induced. For example, iPS cells derived fromfibroblasts may be characterized by down-regulation of the fibroblastcell marker Thy 1 and/or up-regulation of SSEA-3.

Other techniques for nuclear reprogramming which have been reportedinclude nuclear transfer into oocytes (Wakayama et al., Nature394:369-374, 1998; Wilmut et al., Nature 385:810-813, 1997) as well astechniques for nuclear reprogramming of a somatic cell nucleus by fusinga somatic cell and an ES cell (Tada et al., Curr. Biol. 11:1553-1558,2001; Cowan et al., Science 309:1369-73, 2005). Another reportedtechnique for reprogramming a cell nucleus involves treatment of adifferentiated cell with an undifferentiated human carcinoma cellextract (Taranger et al., Mol. Biol. Cell 16:5719-35, 2005).

Induced pluripotent cells can be cultured in any medium used to supportgrowth of pluripotent cells. Typical culture medium includes, but is notlimited to, a defined medium, such as TeSR™. (StemCell Technologies,Inc.; Vancouver, Canada), mTeSR™(StemCell Technologies, Inc.) andStemLine® serum-free medium (Sigma; St. Louis, Mo.), as well asconditioned medium, such as mouse embryonic fibroblast (MEF)-conditionedmedium. As used herein, a “defined medium” refers to a biochemicallydefined formulation comprised solely of biochemically-definedconstituents. A defined medium may also include solely constituentshaving known chemical compositions. A defined medium may further includeconstituents derived from known sources. As used herein, “conditionedmedium” refers to a growth medium that is further supplemented withsoluble factors from cells cultured in the medium. Alternatively, cellscan be maintained on MEFs in culture medium.

Kits

In certain embodiment the invention provides a kit comprising one ormore peptides that bind specifically to a progenitor cell and at leastone container. The kit may optionally comprise one or more progenitorcell lines and instructions for using the kit.

The peptides may be provided lyophilized in a container. Alternatively,the peptides may be provided in a suitable buffer, e.g. PBS. Thepeptides may be linked to one or more detectable substances. The kit mayoptionally comprise one or more nucleic acid sequences encoding for oneor more peptides. The kit may optionally comprise a vector suitable forexpressing the one or more nucleic acid sequences encoding the one ormore peptides. The kit may optionally comprise a cell suitable forexpressing the one or more nucleic acid sequences encoding the one ormore peptides. The progenitor cell lines may be provided frozen in acontainer. A suitable media (e.g. a freezing media) may be provided inthe container with the cells. The kit may optionally comprise a suitablemedia for culturing the progenitor cells. The kit may optionallycomprise one or more antibodies. The antibody may bind specifically toone or more of the peptides provide with the kit. The antibodies mayspecifically bind to one or more proteins expressed by the progenitorcells. The kit may comprise one or more wash solutions. The washsolution may be a suitable buffer, such as PBS. The kit may optionallyprovide a vessel, e.g. such as a multiwell plate, for growing progenitorcells in culture. The kit may optionally comprise one or more containersfor mixing the peptides with the progenitor cells. The kit mayoptionally comprise one or more detectable substances, e.g. quantum dots(Qdots).

The kit may be used to identify peptides that bind to progenitor cells.The kit may be used to screen for progenitor cells that bind to one ormore peptides provided in the kit. The kit may be used to monitor the invitro growth and development (e.g. the differentiation) of one or moreprogenitor cells. The kit may be used to monitor the in vivo growth anddevelopment (e.g. the differentiation) of one or more progenitor cells.The kit may be used to isolate or enrich one or more progenitor celllines.

EXAMPLES Example 1 Cell Culture

The W10 and other embryonic progenitor cell lines were obtained fromBioTime, Inc. (Alameda, Calif.) and human pluripotent stem cells (hEScell line, H9) were obtained from the Stem Cell Core at Sanford-BurnhamMedical Research Institute (La Jolla, Calif.). Embryonic progenitor cellline (P12-30), human dermal fibroblasts (Invitrogen, P2-10) and coronaryartery smooth muscle cells (CASMS) (Lonza, P6-19) were grown followingmanufacturers' instructions. Human embryonic stem cells (P37-55) werecultured as colonies using standard conditions (Leonardo T R, Schell JP, Nickey K S, Tran H T (2012) Chapter 1-Culturing Human PluripotentStem Cells on a Feeder Layer. In: Peterson S, Loring J F, editors. HumanStem Cell Manual, A Laboratory Guide. 2nd ed. Boston: Academic Press.pp. 3-14).

Example 2 Flow Cytometry

For flow cytometric analysis of labeled cells, cells were removed fromthe plates using TrypLE for 5 min at 37° C. Cells were resuspended inPBS, passed through strain top tubes and analyzed using flow cytometer.Control samples included unlabeled cells and cells labeled withuntargeted Qdots. For each sample, 10,000 events were quantified. TheLSRFortessa flow cytometer (BD Biosciences) was used with a violet laserexcitation at 405 nm with a 605/23 bandpass filter to detect Qdot605 andthe yellow laser excitation at 561 nm with a 670/30 bandpass filter todetect Qdot655. Cell autofluorescence was detected with the blue laserexcited at 488 nm and a 510/25 bandpass filter.

Example 3 Gene Expression Analysis

Total RNA was extracted directly from cells growing in 6-well or 6 cmtissue culture plates using Qiagen RNeasy mini kits (Qiagen,Gaithersburg, Md.) according to the manufacturer's instructions. RNAconcentrations were measured using a Beckman DU530 or Nanodropspectrophotometer and RNA quality determined by denaturing agarose gelelectrophoresis or an Agilent 2100 Bioanalyzer. Whole-genome expressionanalysis was carried out using Illumina Human Ref-8v3 BeadArrays and RNAlevels for certain genes were confirmed by quantitative PCR. ForIllumina BeadArrays, total RNA was linearly amplified and biotin-labeledusing Illumina TotalPrep kits (Illumina, San Diego, Calif.), and cRNAwas quality controlled using an Agilent 2100 Bioanalyzer. cRNA washybridized to Illumina BeadChips, processed, and read using aBeadStation array reader according to the manufacturer's instructions(Illumina, San Diego, Calif.). Values less than 90 relative fluorescenceunits (RFUs) were considered nonspecific background signal.

Example 4 Myodifferentiation

W10 cells were grown as micromass cultures by plating 200,000 cells/10μl on 0.1% gelatin-coated wells for 1.5 h before addition of remainingmedia. Micromass cultures were differentiated in myodifferentiationmedia (Smooth Muscle Cell Media 2 and Supplement Mix (PromoCell)+1%GlutaMax+1% penicillin-streptomycin+1 mM pyruvate+10 μMdexamethasone+350 μM l -proline+170 μM l -ascorbic acid+6.25 μg/mlinsulin+6.25 μg/ml transferring+6.25 μg/ml selenious acid+1.25 μg/mlserum albumin+5.35 μg/ml linoleic acid) supplemented with 10 ng/mlTGFβ3).

The embryonic progenitor cell line, W10, expresses markers such astranscription factor, heart and neural crest derivatives-expressed 2(HAND2) and distal HOX genes such as HOXA4 and HOXB7. Whendifferentiated in high density in the presence of 10 ng/ml TGFβ3, acondition that stimulates chondrogenic differentiation of other clonalprogenitor cell lines (4D20.8, SM30, 7PEND24, and E15) and theupregulation of COL2A1 expression (Sternberg et al. (2012) Regen Med 7:481-501), W10 instead displays differentiation to cells with markedlyincreased expression of smooth muscle cell markers such as smooth muscleheavy chain 11 (MYH11) (FIG. 1), calponin 1 (CNN1), myosin light chainkinase (MYLK), and smooth muscle actin (ACTA2) as measured by microarrayanalysis (see, Example 3 above) (FIG. 9).

Example 5 Selection of Cell Binding Peptides from Phage Display Library

Peptide phage display library (Ph.D.-12, New England Biolabs, BeverlyMass.) at 2×10¹¹ pfu was adsorbed against human dermal fibroblasts (HDF)(1×10⁶ cells grown for 48 h in gelatin-coated 10 cm-dish) for 1 h on icein W10 growth media with 2% BSA (library volume: 5 ml). The subtractedlibrary was removed from the HDF and incubated with W10 cells plated ongelatin-coated 10 cm-dish for 2 h at 37° C., with occasional mixing.Cells were washed with washing buffer (1% BSA in PBS+0.9 mMCaCl_(2+0.73) mM MgCl₂) using 100 times the library volume. Cells wereharvested in 1 volume of dissociation buffer (PBS+1 mM EDTA), washedwith 2 volumes of PBS and lysed in 1/15th volume of lysis buffer (30 mMTris pH 7.5+2 mM EDTA+protease inhibitors cocktail (cOmplete, EDTA-freeProtease Inhibitor Cocktail Tablets, Roche Diagnostics) on ice for 1hour. Cells were passed through 25 G needle in 1 ml syringe andinsoluble material was collected by centrifugation at 18000 g for 5 minat 4° C. Cleared lysate was transferred to a clean microcentrifuge tubeand kept on ice until titration and amplification following standardprotocols. In total, three rounds of biopanning were performed usingsimilar conditions, except that the concentration of the amplifiedrecovered phage pool was decreased to 2×10¹⁰ pfu for rounds 2 and 3. Therecovery of the phage pool was calculated as the ratio between therecovered phage and the input phage for each round panning.

Sequencing of Recovered Phage: Individual phage plaques from thetitration plates were grown as individual phage cultures by infection ofthe E. Coli bacteria strain ER2738 (New England Biolabs, Beverly,Mass.). DNA was extracted using the rapid purification of sequencingtemplates protocol (Ph.D. Phage Display Libraries, Manual from NewEngland Biolabs, Version 1.0, 9/09) or amplified by PCR from a peptidephage dilution using primers that hybridize outside the insert (M13KEExt01: 3′ TTGTCATTGTCGGCGCAACT 5′ (SEQ ID NO: 36); M13KE Ext02: 3′GCATTCCACAGACAGCCCTCA 5′ (SEQ ID NO: 37)). DNA was sequenced usingprimer −96 gIII (3′ CCCTCATAGTTAGCGTAACG 5′ (SEQ ID NO: 38)). Thecorresponding peptide sequences were analyzed using the EMBOSS suite ofbioinformatic software and their similarities were identified byClustalW analysis. Homologous peptide sequences were identified inPepBank (available online at pepbank.mgh.harvard.edu) using theSmith-Waterman search algorithm against public peptide library (201572residues in 21672 sequences) and selecting sequences with E( )<1.Homologous protein sequences were identified in the Homo sapiens RefSeqprotein database using Blastp available online.

Results:

Cell targeting peptide phages were selected from a 12-mer linear peptidedisplay library by 3 rounds of selection against undifferentiated W10progenitor cells which included a negative selection against adult humandermal fibroblast (HDF) cells at each round to remove peptides bindingcommon cell surface markers (FIG. 2A). After each round of selection,the percentage of input phage recovered from the target cells increasedindicating enrichment of the phage library for W10 cell binding peptides(FIG. 2B). Peptide sequences were obtained from a sample of 23individual peptide display phages recovered from rounds 2 and 3.Over-representation of several unique sequences and families of relatedsequences indicated a collapse of the library diversity as early asround 2. Sequences from candidate peptide phages were compared usingClustalW multiple sequence alignment software (FIG. 2C). Small peptidemotifs were identified in several families of related sequences (FIG.2D). The rare amino acid, tryptophan, appeared in the second position in7 of the 24 12-mer peptide sequences suggesting selective pressure forbinding to a cell surface epitope. Homology of selected peptides toknown proteins can sometimes be informative for identification ofcandidates for the native cell binding ligand. Short peptide homologiesto intracellular, membrane and extracellular proteins, identified byBLAST searching, did not indicate obvious similarities to functionallyrelevant domains of known cell binding proteins with the possibleexception of plexin homology (FIG. 10A). Both W10-R2-1 and W10-R3-18which have no homology to each other share homology in the extracellulardomains of plexin B1/B3 and plexin B2 respectively, and W10-R3-18 alsoshares homology in the plexin binding domain of semaphorin3C (FIG. 10B).

Example 6 Immunofluorescent Staining of Bound Phage

The binding specificity of selected phages was determined byimmunofluorescent staining of bound phage to the surface of W10progenitor cell line. Cells were plated at 100,000 cells/well in 24 wellplates and incubated overnight. Phages at 2×10¹⁰ pfu/well were dilutedin 0.5 ml of W10 growth media supplemented with 2% BSA and incubatedwith live cells for 2 h at 37° C. Cells were washed as for the selectionexperiments and fixed by 4% paraformaldehyde for 20 min at RT. Cellswere washed twice with PBS and permeabilized by ice-cold MeOH for 15 minon ice. After another two washes, cells were blocked with 5% goat serumin 2% BSA in PBS for 1 h at RT or overnight at 4° C. Cells wereincubated with 1:700 dilution of rabbit anti-Fd bacteriophage antibody(Sigma, B7786) in 2% BSA in PBS for 1 h at RT. Cells were washed with 2ml of 1% BSA in PBS three times, and incubated with 1:1000 dilution ofgoat anti-rabbit AlexaFluor568 conjugated antibody (Invitrogen, A11011)in 1% BSA in PBS for 1 h at RT. Cells were counterstained with DAPI at0.1 μg/ml after final washes and photographed by an Olympus IX71fluorescence microscope. Images were taken and processed using MetaMorph(version 7.5.6.0, Molecular Devices), ImageJ (version 1.45b, NationalInstitute of Health) or Photoshop (version 9.0.2, Adobe) software.

Sixteen of the candidate peptide phages were analyzed (FIG. 2C) forbinding to undifferentiated W10 cells using conditions similar to thatused for phage library selection. The peptide phages with the strongestbinding as detected by immunocytochemical staining (W10 R2 1, W10 R2 11,W10 R2 21 and W10 R3 18) are shown in FIG. 3A. Little or no binding wasdetected for the control M13KE phage (no displayed peptide) or Gly12control phage, which displayed a 12-mer glycine repeat peptide (FIG. 3A)Immunostaining by the 4 peptide phages was stronger than a peptide phagedisplaying a RGD integrin binding peptide, DGARYCRGDCFDG (Holig et al.(2004) Protein Eng Des Sel 17: 433).

Example 7 Binding Factor Determination

Phage binding to W10 progenitor cell line was quantified by titration.Cells were plated at 100,000 cells/well in 24 well plates and incubatedovernight. Phages at 2×10¹⁰ pfu/well were diluted in 0.5 ml of W10growth media supplemented with 2% BSA and incubated with live cells for2 h at 37° C. Cells were washed as for the biopanning experiments; lysisbuffer was added directed to the plated cells (100 μl per well) andincubated for at least 1 h on ice. Cells were scraped from the platewith the aid of a P200 tip, transferred to microcentrifuge tubes andlysate was cleared by centrifugation (18,000 g for 5 min at 4° C.).Cleared lysates were titrated by standard protocols using sequentialdilutions prepared in PBS. Lysate protein concentration was measuredusing the Pierce microBCA assay (Thermo Scientific, Rockford, Ill.)using the 96 well plate format according to manufacturer's instructions.The relative binding factor was calculated as the ratio between therecovery (output/input) per μg of protein for the candidate phage andthe M13KE control phage. Duplicate independent experiments wereperformed for binding factor determination.

Binding of peptide phages to W10 cells was quantified by measuring thepercentage of input phages retained in the cell lysate followingincubation of the cells with the phage at 37° C. The binding factor (BF)was calculated as the ratio of the percentage input recovered for eachcandidate phage to the percentage of input recovered using M13KE controlphage. All 4 peptide phages showed similarly strong W10 binding, withBFs that were statistically different from that of the control phageM13KE; BFs for RGD and Gly12 phages were not significantly differentfrom the M13KE control (FIG. 3B).

Example 8 Peptide Competition for Phage Binding

Cells were plated at 100,000 cells/well in 24 well plates and incubatedovernight. The corresponding synthetic peptide for each peptide phage orcontrol peptides (unrelated or scrambled sequence peptide) werepre-incubated with cells at 5 nM or 5 μM in W10 growth mediasupplemented with 2% BSA for 30 min at 4° C. Peptide phages at 2×10¹⁰pfu/well were added to the peptide dilution and incubated with livecells for 1 h at 4° C. Peptide phage bound to cells was detected byimmunostaining and fluorescence microscopy using anti-phage antibody onfixed and permeabilized cells or quantified by titration of phagesrecovered from cell lysates. The percentage of recovered phage for thecompetition assay was normalized by the recovered phage in theno-peptide control. Duplicate independent experiments were performed forcompetition experiments.

We determined the specificity of peptide phage cell binding for thedisplayed peptide by performing competition experiments with syntheticpeptides to indirectly measure the ability of the free peptide to bindthe surface of W10 cells. The degree of phage binding followingpre-incubation with free peptide was initially assessed byimmunofluorescent phage staining. Competition experiments were performedat 4° C. so that competition for phage binding to the cell surface couldbe detected in the absence of phage internalization. This resulted in areduced phage signal compared to incubation at 37° C. presumably becauseof the limited accumulation of internalized phage at 4° C.Representative images of W10 R2 11 surface bound phage for no peptidecontrol and competing W10-R2-11 peptides (100 μM) are shown in FIG. 4A.The N-terminal FITC-labeled peptide failed to compete with phage forbinding to W10 cells. In contrast, the C-terminal biotinylated peptidesuccessfully competed with the peptide phage. Control competitionexperiments with an unrelated FITC-labeled peptide indicated that thecompetition observed was specific. When C-terminal FITC-labeled versionof the peptide was tested in similar competition experiments, thepeptide was able to compete with the phage for binding to W10 cells(data not shown). These data indicate that a free N-terminus wasnecessary for binding to the same W10 surface molecule that isrecognized by the peptide phages. We therefore performed furthercompetition studies using C-terminal biotinylated peptides that can belinked to a variety of labeling moieties for targeted cell labeling. Forthese experiments the competition for peptide phage binding wasquantified by measuring the percentage of input phages that wererecovered from the cell lysate (FIG. 4B). All 4 W10 selected peptideswere able to compete with the equivalent peptide phages for binding toW10 cells. At concentrations as low as 5 nM, competition by 3 of the 4peptides was statistically significant at p <0.05 (W10-R2-21 was theexception). Higher concentrations of competing peptide (5 μM) resultedin statistically significant competition by all 4 peptides (p<0.05).Scrambled or unrelated peptides did not compete effectively at eitherconcentration (not statistically significant). We demonstratedspecificity of the W10 selected peptides by competition experiments withthe free peptides. The competition experiments demonstrated that freepeptide could compete for peptide phage binding at concentrations as lowas 5 nM indicating that the targeting peptides have high affinity fortheir cognate cell surface antigens. The lack of competition withscrambled peptides indicated that the binding for W10-R2-11, W10-R2-21and W10-R3-18 peptides is sequence specific and not a result ofnon-specific interactions. Failure of the N-terminal FITC labeledpeptides to compete for peptide phage binding indicated the need toreplicate the free N-terminus of the peptides that is present when thepeptides are displayed as fusion to the phage pIII coat protein. Thesedata indicate that cell surface binding of the 4 selected peptides wassequence specific and independent of display on the phage particle.

Example 9 Cell Labeling with Peptide Targeted Qdot Complexes

2 μM of Qdot Streptavidin conjugate (Qdot605 ITK SA, Invitrogen,Q10001MP) were diluted in 100 μl of binding buffer (supplied withQdot605 ITK SA) and incubated with 100-fold excess of biotinylatedpeptide on ice for 1 h. Uncoupled biotinylated peptide was removed fromthe mixture by incubating it with streptavidin magnetic beadsequilibrated in PBS on ice for 30 min; placing the mixture on a magneticstand to separate the beads and removed the complexes in solution.SA-beads were washed with PBS and combined with the recovered complexes.For control reactions, Qdots were incubated with binding buffer andtreated in similar way as the peptide complexes. The concentration ofQdot-peptide complexes was estimated based on the final volumerecovered. To label cells with the Qdot-complexes, 100,000 cells wereplated on gelatin-coated wells of 24 well plates were incubated for 6hours; 5 nM of Qdot-peptides in growth media was added to the cells andincubated for 16 h at 37° C. Cells were imaged after washes with PBS toremove the unbound Qdot-complexes.

We initially attempted to label embryonic progenitor cells usingmonomeric C-terminal FITC labeled peptides. However, the resulting celllabeling was minimal even at concentrations as high as 100 μM. The poorcell labeling could be due to low signal strength and/or limitedinternalization of the monomeric peptide because the same peptidesuccessfully competed with peptide phage for cell binding. Accordingly,we chose to use multivalent peptide targeted Qdots to replicate both thehigh valency and sensitivity obtained using peptide targeted phageparticles. Streptavidin conjugated CdSe-ZnS quantum dots (Qdots) wereused to form complexes with the C-terminal biotinylated peptides. Qdotstypically contain 5-10 streptavidin molecules bound per Qdot each ofwhich can bind up to 4 peptides resulting in multivalent display of20-40 peptides per Qdot. W10 cells were incubated with W10 peptide-Qdotcomplexes and cell labeling detected by fluorescence microscopy.Efficient cell labeling was obtained using overnight incubation atestimated concentrations of 5 nM. These conditions resulted in little orno cell labeling using untargeted Qdots (FIG. 5A).

Competition experiments were used to indirectly determine the ability ofthe targeted Qdot complexes to bind W10 progenitor cells and to comparemultivalent Qdot complexes with monomeric peptides (FIG. 5B). Thepeptide targeted Qdot complexes successfully competed with theequivalent peptide phages for binding to W10 progenitor cells, resultingin a 80% to 95% reduction in cell binding compared to binding in theabsence of competing peptide (p<0.05). Both monomer peptide andmultivalent Qdot complexes competed effectively for peptide phagebinding at 5 nM (>65% inhibition; p<0.05). Competition by any of the 4peptides did not differ significantly from competition by the equivalentpeptide Qdot complex (ANOVA analysis). These data indicate thatdifferences in cell labeling between monomer peptide and multivalentQdots may be the result of more efficient internalization by the peptideQdot complexes rather than differences in binding. The untargeted Qdots,at the same concentration as peptide-Qdot complexes were notstatistically different from the no-peptide control indicating that thepeptide-Q-dot complex competition was dependent on the presence of thepeptide.

Cell targeted Qdots are useful reagents for labeling cells for bothquantitative analysis and cell separation by flow cytometry. With thisapplication in mind, we tested the peptide-Qdot complexes for theirability to label W 10 cells for flow cytometry. Results showed a strongfluorescent shift of W10 cells labeled with W10 R3 18 Qdot complexescompared to cells labeled under similar conditions with untargeted Qdots(FIG. 5C). Using flow cytometric analysis (see Example 2, above), thepercentage of cells that took up the Qdot complexes was determined.Cells treated with untargeted Qdots were used for gating (FIG. 5C). Thepercentage of W10 cells labeled with peptide-Qdot complexes ranged from90% for W10 R3 18 to >75% for W10 R2 21 and W10 R2 11 to 30% for W10 R21 (FIG. 6). These data were consistent with the rank order ofpeptide-Qdot cell labeling observed by fluorescence microscopy.

Peptide selectivity for embryonic progenitor cell lines: The selectivityof the peptides for W10 cells was assessed by comparing targeted Qdotlabeling of W10 cells with 9 other embryonic progenitor cell lines thathave been shown to be distinct cell types by genome expression profiling(West et al. (2008) Regen Med 3: 287. The percentage of cells labeled bythe peptide targeted Qdots was measured by flow cytometry (see Example2, above) (FIG. 6 and Supplemental FIG. 8). All 4 peptides showed somedegree of selective cell targeting. The W10 R3 18 peptide, which wasmost efficient labeling peptide for W10 cells, was the most promiscuouscell targeting peptide. It bound to a high percentage of cells in 7 outof the 10 embryonic progenitor cell lines. The selective labelingprofiles of W10 R2 11 and W10 R2 21 peptides were very similar to eachother, with very little labeling of 2 clonal progenitor lines, 4D20.8and E15, and a high percentage binding of the 7PEND24 cell line. Indeed,these 2 peptides share sequence identity at positions 1 and 2 (G, W),have strongly conserved residues at position 5 and 11 (D/E, M/F) andweakly conserved residues in the last position (R/N). Competitionexperiments showed that these 2 peptides can compete with each other forW10 cell binding (not shown). Taken together, these data suggest thatthe 2 peptides might bind the same cell surface epitope. More restrictedcell labeling was observed with W10 R2 1 Qdot complexes. Of the 10clonal progenitor lines tested, only W10, 7PEND24, SM30 and MW1 celllines showed more than 15% cell labeling and no complex uptake wasobserved for E15 and 4D20.8 cells. The labeling of different embryonicprogenitor cell lines gave an indication of the selectivity of thepeptides complexes. While the binding is not exclusive to the W10 cellline they were selected on, there was a difference in the pattern ofprogenitor cell line targeting depending on the peptide sequence.

Several reports have shown that functionalized Qdots do not cause anydeleterious effects on cell survival in vitro (Slotkin et al. (2007) DevDyn 236: 3393; Jaiswal et al. (2003) Nat Biotechnol 21: 47) and that thedelivery of Qdots by electroporation or lipofection does not disruptearly stages of mammalian development or early embryogenesis noradversely affect embryonic stem cell viability, proliferation ordifferentiation (Lin et al. (2007) BMC Biotechnol 7: 67). Here, we havedemonstrated selective cell labeling using peptide targeted Qdots anddetermined the percentage of labeling of live cells by flow cytometry.The peptide Qdot cell labeling was not exclusive to W10 cells but wasshared to various degrees with other progenitor cell lines with littleor no cell labeling of 2 lines (E15 and 4D20.8). Interestingly, these 2cell lines share a common derivation pathway that is distinct from theother lines (West et al. (2008) Regen Med 3: 287). These data indicatethat the targeting of W10 selected peptide was restricted to certainprogenitor cell types but was not limited the smooth muscle progenitorcell line. A significant advantage of identifying peptides that cantarget Qdots to live cells is that they could potentially be used forlabeling and isolating viable hPS derived differentiating stem cells forfurther culture and characterization. For example, this approach couldbe used to characterize the small fraction of hPS derived mesodermalcells that were labeled by peptide targeted Qdots. The persistence ofthe Qdot signal could also be used for progenitor cell tracking duringdifferentiation of hPS cells to determine cell fate. The peptides couldalso be used to target magnetic particles as an alternative approach forseparating cells using magnetic activated cells sorting which has beenused successfully for preclinical and clinical cell transplantapplications (Grutzkau A, Radbruch A (2010) Cytometry A 77: 643).

Example 10 Differentiation of Cell Lineages Representing 3 Germ Layers

For ectoderm differentiation conditions, embryoid bodies (EBs) wereformed from colonies by manual techniques and grown in complete NPCmedia (DMEM-F12:Neurobasal media 1:1+50 μl/ml BIT9500 Serum substitute(Stemgent)+1% GlutaMax+1% penicillin-streptomycin+1 μl/ml B27 supplement(Invitrogen)+5 mM nicotinamide+5 μg/ml insulin+20 ng/ml EGF+20 ng/mlbFGF) in low attachment plates for 6 days; EBs were then plated onfibronectin-coated wells and grown for another 3 days. For mesodermdifferentiation conditions, EBs were cultured as above except that mediawas DMEM-F12 with GlutaMax+20% FBS+1% NEAA+1% penicillin-streptomycin,and plated on 0.1% gelatin-coated wells. For endodermal differentiationconditions, undifferentiated H9 cells were transferred from coloniesgrowing with MEFs to Geltrex™ (Life Technologies, Grand Island,N.Y.)-coated wells and grown for 2 days with MEF-conditioned media+4ng/ml bFGF. From day 2, cells were grown for another 5 days in RPMI+0.5%FBS+100 ng/ml Activin A.

The cells were analyzed as described in the next example.

Example 11 Immunofluorescent Detection of Differentiation Markers

To confirm lineage commitment of cells differentiated under differentconditions, cells were washed with PBS and fixed with 4% p-formaldehydefor 20 min at RT. After three washes with PBS, cells were permeabilizedand blocked using 5% serum (goat or donkey, depending on primaryantibody)+1% BSA+0.3% Triton X-100 in PBS for 1 h at RT. Primaryantibodies were diluted in 1% BSA+0.3% Triton X-100 in PBS and incubatedovernight 4° C. Antibodies and dilutions used are as follows: nestin(Abcam, Ab22035) at 1:200, α-actinin (Sigma, A7811) at 1:200 dilution,SOX17 (Santa Cruz, sc-17355) at 1:400 dilution, OCT3/4 (R&D Systems,AF1759) at 1:200 dilution, or MYH11 antibody (Biomedical TechnologiesInc., BT-562) at 1:300 dilution. After three washes with PBS, cells wereincubated with secondary antibody dilutions (1:750) in 1% BSA +0.3%Triton X-100 in PBS for 1 h at RT. Antibodies conjugated to AlexaFluor568 were donkey anti-goat (Invitrogen, A11057), goat anti-mouse(Invitrogen, A11004) or donkey anti-rabbit (Invitrogen, A10042)depending on primary antibody. Cells were counterstained with DAPI at0.1 μg/ml for 15 min at RT.

We tested the W10 cell selected peptides for selective targeting ofembryonic progenitors that appear in early differentiating hPS culturesunder 3 different growth conditions that promote differentiation towardectoderm, mesoderm or definitive endoderm (FIG. 7). Importantly,W10-peptide complexes did not label undifferentiated H9 cells indicatingthat they are indeed selective for differentiated cells. Little or nocell labeling was observed when hPS cells were grown under ectodermdifferentiation conditions for any of the 4 different peptide-Qdotscomplexes. Cell labeling was highly restricted, resulting in a few smallpatches of labeled cells, for hPS cells grown under mesodermdifferentiation conditions and incubated with W10 R2 11, W10 R2 21 andW10 R3 18 targeted Qdot complexes but no labeling was observed with W10R2 1 (FIG. 7A). In contrast, a high percentage of cells were labeledwhen hPS cells were differentiated using culture conditions fordefinitive endoderm (high activin A, low serum) and incubated with W10R2 11, W10 R2 21 and W10 R3 18 targeted Qdot complexes. In contrast,cell labeling was highly restricted to a small percentage of cells whenthe endoderm differentiated cells were incubated with W10 R2 1complexes. The hPS cells that were differentiated under the same 3conditions were not labeled by incubation with untargeted Qdotsindicating that in each case the cell labeling was dependent on thetargeting peptide (FIG. 7A). Immunostaining with differentiationspecific markers was used to confirm differentiation toward theappropriate lineage fate (FIG. 7B). Taken together, these data indicatethat the W10 selected cell targeting peptides are capable ofdistinguishing between different types of embryonic progenitor cellswith a marked preference for targeting early definitive endodermalprogenitor cells.

What is claimed is:
 1. A method of enriching a population of progenitorcells, wherein the progenitor cells are the in vitro progeny ofpluripotent stem cells, comprising contacting a population of cellscomprising a progenitor cell with a peptide that binds specifically tothe progenitor cell and separating the progenitor cell bound to thepeptide from the population of cells thereby enriching for a populationof progenitor cells.
 2. The method of claim 1, wherein the peptide ischosen from SWTYSYPNQNMD (SEQ ID NO: 1); DWTYSLPGLVEE (SEQ ID NO: 2);NWTWSMPTGNPA(SEQ ID NO: 3); GMTLRVLTN-YTE- (SEQ ID NO: 4); TLHVSENSWTYN(SEQ ID NO: 5); DWLWSFAPNVDT (SEQ ID NO: 6); TLSSQNPYMHKK (SEQ ID NO:7); IDKQMMTSHKAI (SEQ ID NO: 8); QGMETQKLRMLK (SEQ ID NO: 9);GWYWETPLDMFN (SEQ ID NO: 10); GWVIDYDYYPMR (SEQ ID NO: 11); VTAENYQSFSVS(SEQ ID NO: 12); NNKMDDRMMMSIV (SEQ ID NO: 13); STGTDLHSNARI (SEQ ID NO:14); YEFDNLLNRTLW (SEQ ID NO: 15); EWTVNERTMWDL (SEQ ID NO: 16).
 3. Themethod of claim 1, wherein the progenitor cell is a human cell.
 4. Themethod of claim 3 wherein the progenitor cell is an endoderm cell. 5.The method of claim 3, wherein the progenitor cell is a mesoderm cell.6. The method of claim 3, wherein the progenitor cell is a W10progenitor cell.
 7. The method of claim 3, wherein the progenitor cellexpresses the markers HAND2, HOXA4 and HOXB7.
 8. The method of claim 1,wherein the peptide has plexin homology.